L-ornithine phenyl acetate and methods of making thereof

ABSTRACT

Disclosed herein are forms of L-ornithine phenyl acetate and methods of making the same. A crystalline form may, in some embodiments, be Forms I, II, III and V, or mixtures thereof. The crystalline forms may be formulated for treating subjects with liver disorders, such as hepatic encephalopathy. Accordingly, some embodiments include formulations and methods of administering L-ornithine phenyl acetate.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/037,655, filed Jul. 17, 2018 and to be issued as U.S. Pat. No.10,550,069; which is a continuation of U.S. application Ser. No.15/469,359, filed Mar. 24, 2017, now U.S. Pat. No. 10,173,964; which isa divisional of U.S. application Ser. No. 14/715,481, filed May 18,2015, now U.S. Pat. No. 9,604,909; which is a divisional of U.S.application Ser. No. 14/299,940, filed Jun. 9, 2014, now U.S. Pat. No.9,034,925; which is a divisional of U.S. application Ser. No.13/937,107, filed Jul. 8, 2013, now U.S. Pat. No. 8,785,498; which is adivisional of U.S. application Ser. No. 13/436,642, filed Mar. 30, 2012,now U.S. Pat. No. 8,492,439; which is a divisional of U.S. applicationSer. No. 12/753,763, filed Apr. 2, 2010, now U.S. Pat. No. 8,173,706;which claims the benefit of priority of U.S. Provisional Application No.61/166,676, filed Apr. 3, 2009. The priority documents are herebyincorporated by reference in their entireties. Any and all applicationsfor which a foreign or domestic priority claim is identified in theApplication Data Sheet as filed with the present application, or anycorrection thereto, are hereby incorporated by reference under 37 CFR1.57.

BACKGROUND Field

The present application relates to the fields of pharmaceuticalchemistry, biochemistry, and medicine. In particular, it relates toL-ornithine phenyl acetate salts and methods of making and using thesame.

Description

Hyperammonemia is a hallmark of liver disease and is characterized by anexcess of ammonia in the bloodstream. Hepatic encephalopathy is aprimary clinical consequence of progressive hyperammonemia and is acomplex neuropsychiatric syndrome, which may complicate acute or chronichepatic failure. It is characterized by changes in mental stateincluding a wide range of neuropsychiatric symptoms ranging from minorsigns of altered brain function to overt psychiatric and/or neurologicalsymptoms, or even deep coma. The accumulation of unmetabolized ammoniahas been considered as the main factor involved in the pathogenesis ofhepatic encephalopathy, but additional mechanisms may be associated.

L-Ornithine monohydrochloride and other L-ornithine salts are availablefor their use in the treatment of hyperammonemia and hepaticencephalopathy. For example, U.S. Publication No. 2008/0119554, which ishereby incorporated by reference in its entirety, describes compositionsof L-ornithine and phenyl acetate for the treatment of hepaticencephalopathy. L-ornithine has been prepared by enzymatic conversionmethods. For example, U.S. Pat. Nos. 5,405,761 and 5,591,613, both ofwhich are hereby incorporated by reference in their entirety, describeenzymatic conversion of arginine to form L-ornithine salts. Sodiumphenyl acetate is commercially available, and also available as aninjectable solution for the treatment of acute hyperammonemia. Theinjectable solution is marketed as AMMONUL.

Although salt forms may exhibit improved degradation properties, certainsalts, particularly sodium or chloride salts, may be undesirable whentreating patients having diseases associated with the liver disease,such as hepatic encephalopathy. For example, a high sodium intake may bedangerous for cirrhotic patients prone to ascites, fluid overload andelectrolyte imbalances. Similarly, certain salts are difficult toadminister intravenously because of an increased osmotic pressure, i.e.,the solution is hypertonic. High concentrations of excess salt mayrequire diluting large volumes of solution for intravenousadministration which, in turn, leads to excessive fluid overload.Accordingly, there exists a need for the preparation of L-ornithine andphenyl acetate salts which are favorable for the treatment of hepaticencephalopathy or other conditions where fluid overload and electrolyteimbalance are prevalent.

SUMMARY

Some embodiments disclosed herein include a composition comprising acrystalline form of L-ornithine phenyl acetate.

In some embodiments, the crystalline form exhibits an X-ray powderdiffraction pattern comprising at least one characteristic peak, whereinsaid characteristic peak is selected from the group consisting ofapproximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8° and 24.1° 2θ. In someembodiments, the crystalline form exhibits an X-ray powder diffractionpattern comprising at least three characteristic peaks, wherein saidcharacteristic peaks are selected from the group consisting ofapproximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8° and 24.1° 2θ. In someembodiments, the crystalline form exhibits an X-ray powder diffractionpattern comprising characteristic peaks at approximately 6.0°, 13.9°,14.8°, 17.1°, 17.8° and 24.1° 2θ.

In some embodiments, the crystalline form has a melting point of about202° C. In some embodiments, the crystalline form exhibits a singlecrystal X-ray crystallographic analysis with crystal parametersapproximately equal to the following: unit cell dimensions: a=6.594(2)Å, b=6.5448(18) Å, c=31.632(8) Å, α=90°, β=91.12(3°), γ=90°; CrystalSystem: Monoclinic; and Space Group: P2₁. In some embodiments, thecrystalline form is represented by the formula [C₅H₁₃N₂O₂][C₈H₇O₂].

Some embodiments have the crystalline form exhibit an X-ray powderdiffraction pattern comprising at least one characteristic peak, whereinsaid characteristic peak is selected from the group consisting ofapproximately 4.9°, 13.2°, 17.4°, 20.8° and 24.4° 2θ. In someembodiments, the crystalline form exhibits an X-ray powder diffractionpattern comprising at least three characteristic peaks, wherein saidcharacteristic peaks are selected from the group consisting ofapproximately 4.9°, 13.2°, 17.4°, 20.8° and 24.4° 2θ. In someembodiments, the crystalline form exhibits an X-ray powder diffractionpattern comprising characteristic peaks at approximately 4.9°, 13.2°,17.4°, 20.8° and 24.4° 2θ.

Some embodiments have the crystalline form comprising water and/orethanol molecules. In some embodiments, the crystalline form comprisesabout 11% by weight of said molecules as determined by thermogravimetricanalysis. In some embodiments, the crystalline form is characterized bydifferential scanning calorimetry as comprising an endotherm at about35° C. In some embodiments, the crystalline has a melting point at about203° C.

Some embodiments have the crystalline form exhibiting a single crystalX-ray crystallographic analysis with crystal parameters approximatelyequal to the following: unit cell dimensions: a=5.3652(4) Å, b=7.7136(6)Å, c=20.9602(18) Å, α=90°, β=94.986(6°), γ=90°; Crystal System:Monoclinic; and Space Group: P2₁. In some embodiments, the crystallineform is represented by the formula [C₅H₁₃N₂O₂][C₈H₇O₂]EtOH.H₂O.

Some embodiments have the crystalline form exhibiting an X-ray powderdiffraction pattern comprising at least one characteristic peak, whereinsaid characteristic peak is selected from the group consisting ofapproximately 5.8°, 14.1°, 18.6°, 19.4°, 22.3° and 24.8° 2θ. In someembodiments, the crystalline form exhibits an X-ray powder diffractionpattern comprising at least three characteristic peaks, wherein saidcharacteristic peaks are selected from the group consisting ofapproximately 5.8°, 14.1°, 18.6°, 19.4°, 22.3° and 24.8° 2θ. In someembodiments, the crystalline form exhibits an X-ray powder diffractionpattern comprising characteristic peaks at approximately 5.8°, 14.1°,18.6°, 19.4°, 22.3° and 24.8° 2θ.

In some embodiments, the crystalline form is characterized bydifferential scanning calorimetry as comprising an endotherm at about40° C. In some embodiments, the crystalline form comprises a meltingpoint at about 203° C.

In some embodiments, the crystalline form exhibits an X-ray powderdiffraction pattern comprising at least one characteristic peak, whereinsaid characteristic peak is selected from the group consisting ofapproximately 13.7°, 17.4°, 19.8°, 20.6° and 23.7° 2θ. In someembodiments, the crystalline form exhibits an X-ray powder diffractionpattern comprising at least three characteristic peaks, wherein saidcharacteristic peak is selected from the group consisting ofapproximately 13.7°, 17.4°, 19.8°, 20.6° and 23.7° 2θ. In someembodiments, the crystalline form exhibits an X-ray powder diffractionpattern comprising characteristic peaks at approximately 13.7°, 17.4°,19.8°, 20.6° and 23.7° 2θ.

In some embodiments, the crystalline form is characterized bydifferential scanning calorimetry as comprising an endotherm at about174° C. In some embodiments, the crystalline form has a melting point ofabout 196° C. In some embodiments, the crystalline form comprises apharmaceutically acceptable carrier.

Some embodiments disclosed herein have a composition comprising: atleast about 50% by weight of a crystalline form of L-ornithine phenylacetate salt and at least about 0.01% by weight benzoic acid or a saltthereof.

In some embodiments, the composition comprises at least about 0.10% byweight benzoic acid or a salt thereof. In some embodiments, thecomposition comprises no more than 5% by weight benzoic acid or a saltthereof. In some embodiments, the composition comprises no more than 1%by weight benzoic acid or a salt thereof.

In some embodiments, the composition further comprises at least 10 ppmsilver. In some embodiments, comprises at least 20 ppm silver. In someembodiments, the composition further comprises at least 25 ppm silver.In some embodiments, comprises no more than 600 ppm silver. In someembodiments, composition comprises no more than 100 ppm silver. In someembodiments, the composition comprises no more than 65 ppm silver.

In some embodiments, about 50 mg/mL of the composition in water isisotonic with body fluids. In some embodiments, the isotonic solutionhas an osmolality in the range of about 280 to about 330 mOsm/kg.

In some embodiments, the composition has a density in the range of about1.1 to about 1.3 kg/m³.

Some embodiments disclosed herein include a process for makingL-ornithine phenyl acetate salt comprising: intermixing an L-ornithinesalt, a benzoate salt and a solvent to form an intermediate solution;intermixing phenyl acetate with said intermediate solution; andisolating a composition comprising at least 70% crystalline L-ornithinephenyl acetate by weight.

In some embodiments, the process comprises removing at least a portionof a salt from said intermediate solution before intermixing the phenylacetate, wherein said salt is not an L-ornithine salt. In someembodiments, the process comprises adding hydrochloric acid beforeremoving at least a portion of the salt.

In some embodiments, intermixing the L-ornithine, the benzoate salt andthe solvent comprises: dispersing the L-ornithine salt in water to forma first solution; dispersing the benzoate salt in DMSO to form a secondsolution; and intermixing said first solution and said second solutionto form said solution.

In some embodiments, the composition comprises at least about 0.10% byweight benzoate salt. In some embodiments, composition comprises no morethan 5% by weight benzoate salt. In some embodiments, compositioncomprises no more than 1% by weight benzoate salt.

In some embodiments, the L-ornithine salt is L-ornithine hydrochloride.In some embodiments, the benzoate salt is silver benzoate.

In some embodiments, the composition comprises at least 10 ppm silver.In some embodiments, composition comprises at least 20 ppm silver. Insome embodiments, the composition comprises at least 25 ppm silver. Insome embodiments, the composition comprises no more than 600 ppm silver.In some embodiments, the composition comprises no more than 100 ppmsilver. In some embodiments, the composition comprises no more than 65ppm silver.

In some embodiments, the phenyl acetate is in an alkali metal salt. Insome embodiments, the alkali metal salt is sodium phenyl acetate.

In some embodiments, the composition comprises no more than 100 ppmsodium. In some embodiments, the composition comprises no more than 20ppm sodium.

In some embodiments, the L-ornithine is in a halide salt. In someembodiments, the halide salt is L-ornithine hydrochloride.

In some embodiments, the composition comprises no more than 0.1% byweight chloride. In some embodiments, the composition comprises no morethan 0.01% by weight chloride.

Some embodiments disclosed herein include a composition obtained by anyof the processes disclosed herein.

Some embodiments disclosed herein include a process for makingL-ornithine phenyl acetate salt comprising: increasing the pH value of asolution comprising an L-ornithine salt at least until an intermediatesalt precipitates, wherein said intermediate salt is not an L-ornithinesalt; isolating the intermediate salt from said solution; intermixingphenyl acetic acid with said solution; and isolating L-ornithine phenylacetate salt from said solution.

In some embodiments, the pH value is increased to at least 8.0. In someembodiments, the pH value is increased to at least 9.0. In someembodiments, increasing the pH value comprises adding a pH modifierselected from the group consisting of sodium hydroxide, potassiumhydroxide, sodium methoxide, potassium t-butoxide, sodium carbonate,calcium carbonate, dibutylamine, tryptamine, sodium hydride, calciumhydride, butyllithium, ethylmagnesium bromide or combinations thereof.

Some embodiments disclosed herein include a method of treating orameliorating hyperammonemia in a subject by administering atherapeutically effective amount of a crystalline form of L-ornithinephenyl acetate salt.

In some embodiments, the crystalline form is administered orally.

In some embodiments, the crystalline form is selected from the groupconsisting of Form I, Form II, Form III, Form V, wherein: Form Iexhibits an X-ray powder diffraction pattern having characteristic peaksat approximately 4.9°, 13.2°, 17.4°, 20.8° and 24.4° 2θ; Form IIexhibits an X-ray powder diffraction pattern having characteristic peaksat approximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8° and 24.1° 2θ; Form IIIexhibits an X-ray powder diffraction pattern having characteristic peaksat approximately 5.8°, 14.1°, 18.6°, 19.4°, 22.3° and 24.8° 2θ; and FormV exhibits an X-ray powder diffraction pattern having characteristicpeaks at approximately 13.7°, 17.4°, 19.8°, 20.6° and 23.7° 2θ.

In some embodiments, the crystalline form is Form I. In someembodiments, the crystalline form is Form II. In some embodiments, thecrystalline form is Form III. In some embodiments, the crystalline formis Form V.

In some embodiments, the at least two crystalline forms selected fromthe group consisting of Form I, Form II, Form III and Form V, areadministered. In some embodiments, the at least two crystalline formsare administered at about the same time.

In some embodiments, the crystalline form is administered from 1 to 3times daily. In some embodiments, the therapeutically effective amountis in the range of about 500 mg to about 50 g.

In some embodiments, the subject is identified as having hepaticencephalopathy. In some embodiments, the subject is identified as havinghyperammonemia.

Some embodiments disclosed herein include a process for makingL-ornithine phenyl acetate salt comprising: intermixing an L-ornithinesalt, silver phenyl acetate and a solvent to form a solution, whereinthe L-ornithine salt is a halide salt; and isolating L-ornithine phenylacetate from said solution.

Some embodiments disclosed herein include a method of treating orameliorating hyperammonemia comprising intravenously administering atherapeutically effective amount of a solution comprising L-ornithinephenyl acetate, wherein said therapeutically effective amount comprisesno more than 500 mL of said solution.

In some embodiments, the solution comprises at least about 25 mg/mL ofL-ornithine phenyl acetate. In some embodiments, the solution comprisesat least about 40 mg/mL of L-ornithine phenyl acetate. In someembodiments, the solution comprises no more than 300 mg/mL. In someembodiments, the solution is isotonic with body fluid.

Some embodiments disclosed herein include a method of compressingL-ornithine phenyl acetate, the method comprising applying pressure to ametastable form of L-ornithine phenyl acetate to induce a phase change.

In some embodiments, the metastable form is amorphous. In someembodiments, the metastable form exhibits an X-ray powder diffractionpattern comprising at least one characteristic peak, wherein saidcharacteristic peak is selected from the group consisting ofapproximately 4.9°, 13.2°, 20.8° and 24.4° 2θ.

In some embodiments, the pressure is applied for a predetermined time.In some embodiments, the predetermined time is about 1 second or less.In some embodiments, the pressure is at least about 500 psi.

In some embodiments, the phase change yields a composition having adensity in the range of about 1.1 to about 1.3 kg/m³ after applying thepressure.

In some embodiments, the phase change yields a composition exhibiting anX-ray powder diffraction pattern comprising at least one characteristicpeak, wherein said characteristic peak is selected from the groupconsisting of approximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8° and 24.1°2θ.

Some embodiments disclosed herein include a composition obtained byapplying pressure to a metastable form of L-ornithine phenyl acetate toinduce a phase change.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffraction pattern of Form I.

FIG. 2 shows differential scanning calorimetry results for Form I.

FIG. 3 shows thermogravimetric gravimetric/differential thermal analysisof Form I.

FIG. 4 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of Form I.

FIG. 5 shows dynamic vapor sorption results for Form I.

FIG. 6 is an X-ray powder diffraction pattern of Form II.

FIG. 7 shows differential scanning calorimetry results for Form II.

FIG. 8 shows thermogravimetric gravimetric/differential thermal analysisof Form II.

FIG. 9 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of Form II.

FIG. 10 shows dynamic vapor sorption results for Form II.

FIG. 11 is an X-ray powder diffraction pattern of Form III.

FIG. 12 shows differential scanning calorimetry results for Form III.

FIG. 13 shows thermogravimetric gravimetric/differential thermalanalysis of Form III.

FIG. 14 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of Form III.

FIG. 15 shows dynamic vapor sorption results for Form III.

FIG. 16 is an X-ray powder diffraction pattern of Form V.

FIG. 17 shows differential scanning calorimetry results for Form V.

FIG. 18 shows thermogravimetric gravimetric/differential thermalanalysis of Form V.

FIG. 19 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of Form V.

FIG. 20 shows dynamic vapor sorption results for Form V.

FIG. 21 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of L-ornithine benzoate.

FIG. 22 shows the ¹H nuclear magnetic resonance spectrum obtained from asample of L-ornithine phenyl acetate.

DETAILED DESCRIPTION

Disclosed herein are methods of making L-ornithine phenyl acetate salts,and in particular, crystalline forms of said salt. These methods permitlarge-scale production of pharmaceutically acceptable forms ofL-ornithine phenyl acetate using economical processes. Moreover,crystalline forms of L-ornithine phenyl acetate, including Forms I, II,III and V are also disclosed. The L-ornithine phenyl acetate saltspermit intravenous administration with negligible concomitant sodiumload, and therefore minimize the amount of i.v. fluid that is required.

The present application relates to new crystalline forms of L-ornithinephenyl acetate salts, as well as methods of making and using L-ornithinephenyl acetate salts. The salt advantageously exhibits long-termstability without significant amounts of sodium or chloride. As aresult, L-ornithine phenyl acetate is expected to provide an improvedsafety profile compared to other salts of L-ornithine and phenylacetate. Also, L-ornithine phenyl acetate exhibits lower tonicitycompared to other salts, and therefore can be administered intravenouslyat higher concentrations. Accordingly, L-ornithine phenyl acetate isexpected to provide significant clinical improvements for the treatmentof hepatic encephalopathy.

The present application also relates to various polymorphs ofL-ornithine phenyl acetate. The occurrence of different crystal forms(polymorphism) is a property of some molecules and molecular complexes.Salt complexes, such as L-ornithine phenyl acetate, may give rise to avariety of solids having distinct physical properties like meltingpoint, X-ray diffraction pattern, infrared absorption fingerprint andNMR spectrum. The differences in the physical properties of polymorphsresult from the orientation and intermolecular interactions of adjacentmolecules (complexes) in the bulk solid. Accordingly, polymorphs can bedistinct solids sharing the same active pharmaceutical ingredient yethaving distinct advantageous and/or disadvantageous physico-chemicalproperties compared to other forms in the polymorph family.

Method of Making L-Ornithine Phenyl Acetate Salt

Some embodiments disclosed herein include a method of making L-ornithinephenyl acetate salt. L-Ornithine phenyl acetate may be produced, forexample, through an intermediate salt, such as L-ornithine benzoate. Asshown in Scheme 1, an L-ornithine salt of Formula I can be reacted witha benzoate salt of Formula II to obtain the intermediate L-ornithinebenzoate.

Various salts of L-ornithine may be used in the compound of Formula I,and therefore X in Formula I can be any ion capable of forming a saltwith L-ornithine other than benzoic acid or phenyl acetic acid. X can bea monoatomic anion, such as, but not limited to, a halide (e.g.,fluoride, chloride, bromide, and iodide). X can also be a polyatomicanion, such as, but not limited to, acetate, aspartate, formate,oxalate, bicarbonate, carbonate, bitrate, sulfate, nitrate,isonicotinate, salicylate, citrate, tartrate, pantothenate, bitartrate,ascorbate, succinate, maleate, gentisinate, fumarate, gluconate,glucaronate, saccharate, formate, glutamate, methanesulfonate,ethanesulfonate, benzensulfonate, p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate), phosphate and the like. Insome embodiments, X is a monovalent ion. In some embodiments, X ischloride.

Similarly, the benzoate salt of Formula II is not particularly limited,and therefore Y in Formula II can be any appropriate ion capable offorming a salt with benzoic acid. In some embodiments, Y can be amonoatomic cation, such as an alkali metal ion (e.g., Li⁺, Na⁺, and K⁺)and other monovalent ions (e.g., Ag⁺). Y may also be a polyatomiccation, such as ammonium, L-arginine, diethylamine, choline,ethanolamine, 1H-imidazole, trolamine, and the like. In someembodiments, Y is an inorganic ion. In some embodiments, Y is silver.

Many other possible salts of L-ornithine and benzoic acid may be usedfor the compounds of Formulae I and II, respectively, and can readily beprepared by those skilled in the art. See, for example, Bighley L. D.,et al., “Salt forms of drugs and absorption,” In: Swarbrick J., HorlanJ. C., eds. Encyclopedia of pharmaceutical technology, Vol. 12. NewYork: Marcel Dekker, Inc. pp. 452-499, which is hereby incorporated byreference in its entirety.

The intermediate L-ornithine benzoate (i.e., Formula III) can beprepared by intermixing solutions including compounds of Formulae I andII. As an example, the compounds of Formulae I and II may be separatelydissolved in water and dimethyl sulfoxide (DMSO), respectively. The twosolutions may then be intermixed so that the L-ornithine and benzoicacid react to form the salt of Formula III. Alternatively, the two saltcompounds can be directly dissolved into a single solution. In someembodiments, L-ornithine and benzoic acid are dissolved in separatesolvents, and subsequently intermixed. In some embodiments, L-ornithineis dissolved in an aqueous solution, benzoic acid is dissolved in anorganic solvent, and the L-ornithine and benzoic acid solutions aresubsequently intermixed.

Non-limiting examples of solvents which may be used when intermixingL-ornithine and benzoate salts include acetonitrile, dimethylsulfoxide(DMSO), cyclohexane, ethanol, acetone, acetic acid, 1-propanol,dimethylcarbonate, N-methyl-2-pyrrolidone (NMP), ethyl acetate (EtOAc),toluene, isopropyl alcohol (IPA), diisopropoyl ether, nitromethane,water, 1,4 dioxane, tdiethyl ether, ethylene glycol, methyl acetate(MeOAc), methanol, 2-butanol, cumene, ethyl formate, isobutyl acetate,3-methyl-1-butanol, anisole, and combinations thereof. In someembodiments, the L-ornithine benzoate solution includes water. In someembodiments, the L-ornithine benzoate solution includes DMSO.

Upon intermixing L-ornithine and benzoate salts, counterions X and Y mayform a precipitate that can be removed from the intermixed solutionusing known methods, such as filtration, centrifugation, and the like.In some embodiments, X is chloride, Y is silver, and the reactionproduces a precipitate having AgCl. Although Scheme 1 shows thecompounds of Formulae I and II as salts, it is also within the scope ofthe present application to intermix the free base of L-ornithine andbenzoic acid to form the intermediate of L-ornithine benzoate.Consequently, forming and isolating the precipitate is optional.

The relative amount of L-ornithine and benzoate salts that areintermixed is not limited; however the molar ratio of L-ornithine tobenzoic acid may optionally be in the range of about 10:90 and 90:10. Insome embodiments, the molar ratio of L-ornithine benzoate can be in therange of about 30:70 and 30:70. In some embodiments, the molar ratio ofL-ornithine to benzoate can be in the range of about 40:60 and 60:40. Insome embodiments, the molar ratio of L-ornithine to benzoate is about1:1.

In embodiments where X and Y are both inorganic ions (e.g., X and Y arechloride and silver, respectively), additional amounts of X-containingsalt may be added to encourage further precipitation of the counterionY. For example, if X is chloride and Y is silver, the molar ratio ofL-ornithine hydrochloride to silver benzoate may be greater than 1:1 sothat an excess of chloride is present relative to silver. Accordingly,in some embodiments, the molar ratio of L-ornithine to benzoic acid isgreater than about 1:1. Nevertheless, the additional chloride salt isnot required to be derived from an L-ornithine salt (e.g., L-ornithinehydrochloride). For example, dilute solutions of hydrochloric acid maybe added to the solution to further remove silver. Although it is notparticularly limited when the additional X-containing salt is added, itis preferably added before the AgCl is initially isolated.

As shown in Scheme 2, the L-ornithine benzoate can be reacted with aphenyl acetate salt of Formula IV to form L-ornithine phenyl acetate.For example, sodium phenyl acetate can be intermixed with a solution ofL-ornithine benzoate to form L-ornithine phenyl acetate. Various saltsof phenyl acetate may be used, and therefore Z in Formula IV can be anycation capable of forming a salt with phenyl acetate other than benzoicacid or L-ornithine. In some embodiments, Z can be a monoatomic cation,such as an alkali metal ion (e.g., Li⁺, Na⁺, and K⁺) and othermonovalent ions (e.g., Ag⁺). Z may also be a polyatomic cation, such asammonium, L-arginine, diethylamine, choline, ethanolamine, 1H-imidazole,trolamine, and the like. In some embodiments, Z is an inorganic ion. Insome embodiments, Z is sodium.

The relative amount of L-ornithine and phenyl acetate salts that areintermixed is also not limited; however the molar ratio of L-ornithineto phenyl acetate may optionally be in the range of about 10:90 and90:10. In some embodiments, the molar ratio of L-ornithine to phenylacetate can be in the range of about 30:70 and 30:70. In someembodiments, the molar ratio of L-ornithine to phenyl acetate can be inthe range of about 40:60 and 60:40. In some embodiments, the molar ratioof L-ornithine to benzoic acid is about 1:1.

The L-ornithine phenyl acetate of Formula V may then be isolated fromsolution using known techniques. For example, by evaporating any solventuntil the L-ornithine phenyl acetate crystallizes, or alternatively bythe adding an anti-solvent miscible in the L-ornithine phenyl acetatesolution until the L-ornithine phenyl acetate precipitates fromsolution. Another possible means for isolating the L-ornithine phenylacetate is to adjust the temperature of the solution (e.g., lower thetemperature) until the L-ornithine phenyl acetate precipitates. As willbe discussed in further detail in a later section, the method ofisolating the L-ornithine phenyl acetate affects the crystalline formthat is obtained.

The isolated L-ornithine phenyl acetate may be subjected to variousadditional processing, such as drying and the like. In some embodiments,L-ornithine phenyl acetate may be subsequently intermixed with a diluteHCl solution to precipitate residual silver. The L-ornithine phenylacetate may again be isolated from solution using similar methodsdisclosed above.

As would be appreciated by a person of ordinary, guided by the teachingsof the present application, L-ornithine phenyl acetate may similarly beprepared using an intermediate salt other than L-ornithine benzoate.Thus, for example, L-ornithine, or a salt thereof (e.g., L-ornithinehydrochloride), can be intermixed with a solution having acetic acid.L-Ornithine acetate may then be intermixed with phenyl acetic acid, or asalt thereof (e.g., sodium phenyl acetate), to obtain L-ornithine phenylacetate. Scheme 4 illustrates an exemplary process of formingL-ornithine phenyl acetate using L-ornithine acetate as an intermediatesalt. In some embodiments, the intermediate salt can be apharmaceutically acceptable salt of L-ornithine. For example, theintermediate L-ornithine salt can be an acetate, aspartate, formate,oxalate, bicarbonate, carbonate, bitrate, sulfate, nitrate,isonicotinate, salicylate, citrate, tartrate, pantothenate, bitartrate,ascorbate, succinate, maleate, gentisinate, fumarate, gluconate,glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzensulfonate, p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate) or phosphate. The free acidof the intermediate is preferably a weaker acid relative to phenylacetic acid. In some embodiments, the intermediate is an L-ornithinesalt with an anion component that exhibits a pK_(a) value that is higherthan the pK_(a) value of phenyl acetic acid. As an example, forL-ornithine acetate, acetic acid and phenyl acetic acid exhibit pK_(a)values of about 4.76 and 4.28, respectively.

L-Ornithine phenyl acetate may also be prepared, in some embodiments,without forming an intermediate salt, such as L-ornithine benzoate.Scheme 4 illustrates an exemplary process for preparing L-ornithinephenyl acetate without an intermediate salt. A pH modifier may be addedto a solution of L-ornithine salt (e.g., as illustrated in Scheme 4 bythe compound of Formula I) until a salt precipitates from solution,where the salt is not an L-ornithine salt. As an example, sodiummethoxide (NaOMe) can be added to a solution of L-ornithinehydrochloride until sodium chloride precipitates from solution to leavea free base of L-ornithine. The precipitate may optionally be isolatedfrom solution using known techniques, such as filtration,centrifugation, and the like. The free base of L-ornithine (e.g., asillustrated in Scheme 4 by the compound of Formula I-a) may beintermixed with phenyl acetic acid, or a salt thereof (e.g., asillustrated in Scheme 4 by the compound of Formula IV), to obtainL-ornithine phenyl acetate. The L-ornithine phenyl acetate of Formula Vmay then be isolated as previously described.

A pH modifier can include a basic compound, or anhydrous precursorthereof, and/or a chemically protected base. Non-limiting examples of pHmodifiers include sodium hydroxide, potassium hydroxide, sodiummethoxide, potassium t-butoxide, sodium carbonate, calcium carbonate,dibutylamine, tryptamine, sodium hydride, calcium hydride, butyllithium,ethylmagnesium bromide and combinations thereof. Also, the amount of pHmodifier to be added is not particularly limited; however the molarratio of L-ornithine to pH modifier may optionally be in the range ofabout 10:90 and 90:10. In some embodiments, the molar ratio ofL-ornithine to pH modifier can be in the range of about 30:70 and 30:70.In some embodiments, the molar ratio of L-ornithine to pH modifier canbe in the range of about 40:60 and 60:40. In some embodiments, the molarratio of L-ornithine to pH modifier is about 1:1. The pH modifier may,in some embodiments be added to adjust the pH value to at least about8.0; at least about 9.0; or at least about 9.5.

Another process for forming L-ornithine phenyl acetate, in someembodiments, includes reacting an alkali metal salt of L-ornithine witha phenyl acetate salt. As an example, L-ornithine hydrochloride may beintermixed with silver phenyl acetate and a solvent. AgCl may thenprecipitate and is optionally isolated from the solution. The remainingL-ornithine phenyl acetate can also be isolated using known methods.This process can be completed using generally the same procedures andconditions outlined above. For example, the relative molar amounts ofL-ornithine to phenyl acetate can be 10:90 to 90:10; 30:70 to 70:30;40:60 to 60:40; or about 1:1. Also, the L-ornithine phenyl acetate maybe isolated by evaporating the solvent, adding an anti-solvent, and/orreducing the temperature.

Compositions of L-Ornithine Phenyl Acetate

Also disclosed herein are compositions of L-ornithine phenyl acetate.The compositions of the present application advantageously have lowamounts of inorganic salts, particularly alkali metal salts and/orhalide salts, and therefore are particularly suited for oral and/orintravenous administration to patients with hepatic encephalopathy.Meanwhile, these compositions may exhibit similar stability profilescompared to other salts (e.g., mixtures of L-ornithine hydrochloride andsodium phenyl acetate). The compositions may, in some embodiments, beobtained by one of the processes disclosed in the present application.For example, any of the disclosed processes using L-ornithine benzoateas an intermediate may yield the compositions of the presentapplication.

The compositions, in some embodiments, can include a crystalline form ofL-ornithine phenyl acetate (e.g., Forms I, II, III and/or V disclosedherein). In some embodiments, the composition may include at least about20% by weight of a crystalline form of L-ornithine phenyl acetate(preferably at least about 50% by weight, and more preferably at leastabout 80% by weight). In some embodiments, the composition consistsessentially of a crystalline form of L-ornithine phenyl acetate. In someembodiments, the composition includes a mixture of at least two (e.g.,two, three or four forms) of Forms I, II, III, and V.

The compositions, in some embodiments, include Form II. For example, thecompositions may include at least about 20%; at least about 50%; atleast about 90%; at least about 95%; or at least about 99% of Form II.Similarly, the compositions may also include, for example, Forms I, IIIor V. The compositions may optionally include at least about 20%; atleast about 50%; at least about 90%; at least about 95%; or at leastabout 99% of Forms I, II, III and/or V.

Also within the scope of the present application are amorphous forms ofL-ornithine phenyl acetate. Various methods are known in the art forpreparing amorphous forms. For example, a solution of L-ornithine phenylacetate may be dried under vacuum by lyophilization to obtain anamorphous composition. See P.C.T. Application WO 2007/058634, whichpublished in English and designates the U.S., and is hereby incorporatedby reference for disclosing methods of lyophilization.

It is preferred that the composition have low amounts (if any) of alkaliand halogen ions or salts, particular sodium and chloride. In someembodiments, the composition comprises no more than about 100 ppm ofalkali metals (preferably no more than about 20 ppm, and most preferablyno more than about 10 ppm). In some embodiments, the compositioncomprises no more than about 100 ppm of sodium (preferably no more thanabout 20 ppm, and most preferably no more than about 10 ppm). In someembodiments, the composition comprises no more than about 0.1% by weightof halides (preferably no more than about 0.01% by weight). In someembodiments, the composition comprises no more than about 0.1% by weightof chloride (preferably no more than about 0.01% by weight).

The reduced content of alkali metals and halides provides a compositionsuitable for preparing concentrated isotonic solutions. As such, thesecompositions can be more easily administered intravenously compared to,for example, administering mixtures of L-ornithine hydrochloride andsodium phenyl acetate. In some embodiments, an about 45 to about 55mg/mL solution of L-ornithine phenyl acetate in water (preferably about50 mg/mL) is isotonic with body fluids (e.g., the solution exhibits anosmolality in the range of about 280 to about 330 mOsm/kg).

The compositions may also include residual amounts of the anion from anintermediate salt formed during the process of making the L-ornithinephenyl acetate composition. For example, some of the processes disclosedherein yield compositions having benzoic acid or a salt thereof. In someembodiments, the composition comprises at least about 0.01% by weightbenzoic acid or a salt thereof (preferably at least about 0.05% byweight, and more preferably about 0.1% by weight). In some embodiments,the composition comprises no more than about 3% by weight benzoic acidor a salt thereof (preferably no more than about 1% by weight, and morepreferably no more than about 0.5% by weight). In some embodiments, thecomposition includes a salt, or an acid thereof, in the range of about0.01% to about 3% by weight (preferably about 0.1% to about 1%), whereinthe salt is selected from acetate, aspartate, formate, oxalate,bicarbonate, carbonate, bitrate, sulfate, nitrate, isonicotinate,salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzensulfonate, p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate) or phosphate.

Similarly, a composition prepared using an acetate intermediate may haveresidual amounts of acetic acid or acetate. In some embodiments, thecomposition includes at least about 0.01% by weight acetic acid oracetate (preferably at least about 0.05% by weight, and more preferablyabout 0.1% by weight). In some embodiments, the composition includes nomore than about 3% by weight acetic acid or acetate (preferably no morethan about 1% by weight, and more preferably no more than about 0.5% byweight).

The compositions may also include low amounts of silver. Exemplaryprocesses disclosed herein utilize, for example, silver benzoate, butstill yield compositions with surprisingly low amounts of silver. Thus,in some embodiments, the composition includes no more than about 600 ppmsilver (preferably no more than about 100 ppm, and more preferably nomore than about 65 ppm). In some embodiments, the composition includesat least about 10 ppm silver (alternatively at least about 20 or 25 ppmsilver).

Pharmaceutical Compositions

The compositions of L-ornithine phenyl acetate of the presentapplication may also be formulated for administration to a subject(e.g., a human). L-Ornithine phenyl acetate, and accordingly thecompositions disclosed herein, may be formulated for administration witha pharmaceutically acceptable carrier or diluent. L-ornithine phenylacetate may thus be formulated as a medicament with a standardpharmaceutically acceptable carrier(s) and/or excipient(s) as is routinein the pharmaceutical art. The exact nature of the formulation willdepend upon several factors including the desired route ofadministration. Typically, L-ornithine phenyl acetate is formulated fororal, intravenous, intragastric, subcutaneous, intravascular orintraperitoneal administration.

The pharmaceutical carrier or diluent may be, for example, water or anisotonic solution, such as 5% dextrose in water or normal saline. Solidoral forms may contain, together with the active compound, diluents,e.g. lactose, dextrose, saccharose, cellulose, corn starch or potatostarch; lubricants, e.g. silica, talc, stearic acid, magnesium orcalcium stearate, and/or polyethylene glycols; binding agents, e.g.starches, gum arabic, gelatin, methylcellulose, carboxymethylcelluloseor polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginicacid, alginates or sodium starch glycolate; effervescing mixtures;dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates,laurylsulphates; and, in general, non-toxic and pharmacologicallyinactive substances used in pharmaceutical formulations. Suchpharmaceutical preparations may be manufactured in known manners, forexample, by means of mixing, granulating, tabletting, sugar-coating, orfilm-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions orsuspensions. The syrups may contain as carriers, for example, saccharoseor saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain a carrier, for example a naturalgum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol. The suspensions orsolutions for intramuscular injections may contain, together withL-ornithine phenyl acetate, a pharmaceutically acceptable carrier, e.g.sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol,and if desired, a suitable amount of lidocaine hydrochloride.

The medicament may consist essentially of L-ornithine phenyl acetate anda pharmaceutically acceptable carrier. Such a medicament thereforecontains substantially no other amino acids in addition to L-ornithineand phenyl acetate. Furthermore, such a medicament containsinsubstantial amounts of other salts in addition to L-ornithine phenylacetate.

Oral formulations may generally include dosages of L-ornithine phenylacetate in the range of about 500 mg to about 100 g. Accordingly, insome embodiments, the oral formulation includes the L-ornithine phenylacetate compositions disclosed herein in the range of about 500 mg toabout 50 g. In some embodiments, the oral formulation is substantiallyfree of alkali metal salts and halides (e.g., contains no more thantrace amounts of alkali metal salts and halides).

Intravenous formulations may also generally include dosages ofL-ornithine phenyl acetate in the range of about 500 mg to about 100 g(preferably about 1 g to about 50 g). In some embodiments, theintravenous formulation is substantially free of alkali metal salts andhalides (e.g., contains no more than trace amounts of alkali metal saltsand halides). In some embodiments, the intravenous formulation has aconcentration of about 5 to about 300 mg/mL of L-ornithine phenylacetate (preferably about 25 to about 200 mg/mL, and more preferablyabout 40 to about 60 mg/mL).

The composition, or medicament containing said composition, mayoptionally be placed is sealed packaging. The sealed packaging mayreduce or prevent moisture and/or ambient air from contacting thecomposition or medicament. In some embodiments, the packaging includes ahermetic seal. In some embodiments, the packaging sealed under vacuum orwith an inert gas (e.g., argon) within the sealed package. Accordingly,the packaging can inhibit or reduce the rate of degradation for thecomposition or medicament stored within the packaging. Various types ofsealed packaging are known in the art. For example, U.S. Pat. No.5,560,490, is hereby incorporate by reference in its entirety, disclosesan exemplary sealed package for medicaments.

Compositions with Improved Density

Applicants have surprisingly found that compositions with greaterdensity may be obtained by applying sufficient pressure to compositionshaving Form I (described below) to induce a transition to Form II(described below). For example, applying 3 tons of force for 90 minutesto Forms I and II yield densities of 1.197 kg/m³ and 1.001 kg/m³,respectively. Surprisingly, Form I converted to Form II under theseconditions; therefore the greater density appears to be explained by thedifferent crystalline form as the starting material.

Accordingly, disclosed herein are methods of increasing the density ofan L-ornithine phenyl acetate composition having Form I by applyingpressure to the composition sufficient to induce a transition to FormII. The appropriate amount of force or pressure to induce the phasechange may vary with the amount of time the force or pressure isapplied. Thus, a person of ordinary skill, guided by the teachings ofthe present application, can determine appropriate amounts of pressureand time to induce the phase change. In some embodiments, at least about1 ton of force is applied (preferably at least about 2 tons, and morepreferably about 3 tons). In some embodiments, at least about 500 psi ofpressure is applied (preferably at least about 1000 psi, and morepreferably at least about 2000 psi).

The amount of time for applying pressure is not particularly limited,and as discussed above, will vary depending upon the amount time. Forexample, when applying large forces (e.g., 10 tons) to a typicaltablet-sized punch, the time may be about 1 second or less. In someembodiments, the time for apply pressure is a predetermined time. Thetime may be, for example, about 0.1 seconds; about 1 second; at leastabout 1 minute; at least about 5 minutes; or at least about 20 minutes.

In some embodiments, the composition includes at least about 10% byweight of Form I. In some embodiments, the composition includes at leastabout 30% by weight of Form I.

Without being bound to any particular theory, Applicants believe thegreater density may result, at least in part, from ethanol solvatecomponent present in Form I. Applying pressure to the solvate mayfacilitate forming a denser structure with fewer defects (e.g., grainboundaries). Consequently, in some embodiments, methods of increasingthe density of an L-ornithine phenyl acetate composition having solvatecomponents include applying pressure to the composition sufficient toinduce a transition to Form II. In some embodiments, the pressure is atleast about 500 psi (preferably at least about 1000 psi, and morepreferably at least about 2000 psi). In some embodiments, the time forapply pressure is a predetermined time. In some embodiments, thecomposition includes at least about 10% of the solvate form (preferablyat least about 30%, and more preferably at least about 50%).

The compositions of L-ornithine phenyl acetate disclosed herein maytherefore have higher densities compared to compositions obtain by, forexample, precipitating a crystalline form. In some embodiments, thecomposition has a density of at least about 1.1 kg/m³ (preferably atleast about 1.15 kg/m³, and more preferably at least about 1.18 kg/m³).In some embodiments, the composition has a density of no more than about1.3 kg/m³ (preferably no more than about 1.25 kg/m³, and more preferablyno more than about 1.22 kg/m³). In some embodiments, the composition hasa density of about 1.2 kg/m³.

Crystalline Forms of L-Ornithine Phenyl Acetate

Also disclosed herein are crystalline forms of L-ornithine phenylacetate, and in particular, crystalline Form I, Form II, Form III, andForm V. L-Ornithine phenyl acetate may, in some embodiments, be obtainedusing the processes disclosed above and then crystallized using any ofthe methods disclosed herein.

Form I

The precise conditions for forming crystalline Form I may be empiricallydetermined and it is only possible to give a number of methods whichhave been found to be suitable in practice.

Thus, for example, crystalline Form I may generally be obtained bycrystallizing L-ornithine phenyl acetate under controlled conditions. Asan example, precipitating L-ornithine phenyl acetate from a saturatedsolution by adding ethanol at reduced temperatures (e.g., 4° or −21°C.). Exemplary solvents for the solution that yield crystalline Form Iupon adding ethanol include, but are not limited to, cyclohexanone,1-propanol, dimethylcarbonate, N-methylpyrrolidine (NMP), diethyl ether,2-butanol, cumene, ethyl formate, isobutyl acetate, 3-methyl-1-butanol,and anisole.

Accordingly, in the context of the processes for making L-ornithinephenyl acetate disclosed above, the process can yield Form I byutilizing particular isolation methods. For example, L-ornithine phenylacetate may be isolated by adding ethanol at reduced temperature toyield Form I.

Crystalline Form I was characterized using various techniques which aredescribed in further detail in the experimental methods section. FIG. 1shows the crystalline structure of Form I as determined by X-ray powderdiffraction (XRPD). Form I, which may be obtained by the methodsdisclosed above, exhibits characteristic peaks at approximately 4.9°,13.2°, 17.4°, 20.8° and 24.4° 2θ. Thus, in some embodiments, acrystalline form of L-ornithine phenyl acetate has one or morecharacteristic peaks (e.g., one, two, three, four or five characteristicpeaks) selected from approximately 4.9°, 13.2°, 17.4°, 20.8°, and 24.4°2θ.

As is well understood in the art, because of the experimentalvariability when X-ray diffraction patterns are measured on differentinstruments, the peak positions are assumed to be equal if the two theta(2θ) values agree to within 0.2° (i.e., ±0.2°). For example, the UnitedStates Pharmacopeia states that if the angular setting of the 10strongest diffraction peaks agree to within ±0.2° with that of areference material, and the relative intensities of the peaks do notvary by more than 20%, the identity is confirmed. Accordingly, peakpositions within 0.2° of the positions recited herein are assumed to beidentical.

FIG. 2 shows results obtained by differential scanning calorimetry (DSC)for Form I. These results indicate an endotherm at 35° C., which ispossibly associated with a desolvation and/or dehydration to Form II. Asecond transition at about 203° C. indicates the melting point for thecrystal. To explore the possible existence of a desolvation and/ordehydration transition, Form I was analyzed by thermogravimetricgravimetric/differential thermal analysis (TG/DTA), which is shown inFIG. 3. Form I exhibits a 11.28% weight loss at about 35° C., andtherefore these results further suggest that Form I exhibits adesolvation and/or dehydration transition at about 35° C. The meltingpoint of about 203° C. could also be observed by TGA testing.Accordingly, in some embodiments, the crystalline form of L-ornithinephenyl acetate is characterized by differential scanning calorimetry ashaving an endotherm at about at about 35° C. In some embodiments, acrystalline form of L-ornithine phenyl acetate exhibits a weight loss ofabout 11% at about 35° C., as determined by TGA. In some embodiments, acrystalline form of L-ornithine phenyl acetate exhibits a melting pointof about 203° C.

FIG. 4 shows nuclear magnetic resonance (NMR) integrals and chemicalshifts for Form I. The integrals confirm the presence of L-ornithinephenyl acetate: 7.5 (aromatic CH), 3.8 (CH adjacent to NH₂), 3.6 (CH₂unit of phenyl acetate), 3.15 (CH₂ adjacent to NH₂) and 1.9 (aliphaticCH₂ units) ppm (integrals: 5:1:2:2:4 protons; 1.2, 0.25, 0.5, 0.5, 1.0).Amine protons and hydroxyl protons were not observed due to protonexchange at both the zwitterion and site of salt formation. Meanwhile,FIG. 5 shows dynamic vapor sorption (DVS) results for Form I, and show awater uptake of about 0.2% by weight. XRPD results following DVAanalysis (not shown) confirm that Form I did not transition to adifferent polymorph. Form I can therefore be characterized asnon-hygroscopic and stable over a wide range of humidity.

A 7-day stability study of Form I at 40° C./75% RH indicated that atransformation to Form II occurred under these conditions. Form I alsoconverts to Form II at elevated temperatures (e.g., 80° or 120° C.),with or without applying a vacuum, after 7 or 14 days. Accordingly, FormI is metastable.

Single crystal x-ray diffraction (SXRD) was also used to determine thestructure of Form I at −20° and −123° C., and the results are summarizedin TABLES 1 and 2. The results confirm that Form I is a solvate havingethanol and water molecules within the unit cell. In some embodiments, acrystalline form of L-ornithine phenyl acetate can be represented by theformula C₁₅H₂₈N₂O₆. In some embodiments, a crystalline form ofL-ornithine phenyl acetate can be represented by the formula[C₅H₁₃N₂O₂][C₈H₇O₂]EtOH.H₂O. In some embodiments, a crystalline form ofL-ornithine phenyl acetate exhibits a single crystal X-raycrystallographic analysis with crystal parameters approximately equal tothe following: unit cell dimensions of a=5.3652(4) Å, b=7.7136(6) Å,c=20.9602(18) Å, α=90°, β=94.986(6°), γ=90°; a monoclinic crystalsystem, and a P2₁ space group.

TABLE 1 Crystallographic Data of Form I Collected at −20° C. EmpiricalFormula C₁₅ H₂₈ N₂ O₆ or [C₅H₁₃N₂O₂][C₈H₇O₂]EtOH•H₂O Formula Weight332.39 Crystal System Monoclinic Space Group P2₁ Unit Cell Dimensions a= 5.3652(4) Å α = 90° b = 7.7136(6) Å β = 94.986(6)° c = 20.9602(18) Å γ= 90° Volume 864.16(12) Å³ Number of Reflections 1516 (2.5° < θ < 28°)Density (calculated) 1.277 mg/cm³

TABLE 2 Crystallographic Data of Form I Collected at −123° C. EmpiricalFormula C₁₅ H₂₈ N₂ O₆ or [C₅H₁₃N₂O₂][C₈H₇O₂]EtOH•H₂O Formula Weight332.39 Crystal System Monoclinic Space Group P2₁ Unit Cell Dimensions a= 5.3840(9) Å α = 90° b = 7.7460(12) Å β = 95.050(12)° c = 21.104(4) Å γ= 90° Volume 876.7(3) Å³ Number of Reflections 1477 (2.5° < θ < 18°)Density (calculated) 1.259 mg/cm³Form II

The precise conditions for forming crystalline Form II may beempirically determined and it is only possible to give a number ofmethods which have been found to be suitable in practice.

Thus, for example, crystalline Form II may be prepared bycrystallization under controlled conditions. Crystalline Form II can beprepared by, for example, evaporating a saturated organic solution ofL-ornithine phenyl acetate. Non-limiting examples of organic solutionsthat may be used to obtain Form II include ethanol, acetone,benzonitrile, dichloromethane (DCM), dimethyl sulfoxide (DMSO), ethylacetate (EtOAc), acetonitrile (MeCN), methyl acetate (MeOAc),nitromethane, tert-butyl methyl ether (TBME), tetrahydrofuran, andtoluene. Other solvents may yield a mixture of Form I and II, such as,but not limited to, 1,4 dioxane, 1-butanol, cyclohexane, IPA, THF, MEK,MeOAc and water.

Form II can also be obtained by precipitating L-ornithine phenyl acetatefrom a saturated organic solution by adding an anti-solvent forL-ornithine phenyl acetate, such as IPA. Form II may be precipitatedover a broad range of temperatures (e.g., room temperature, 4° C., and−21° C.). Non-limiting examples of suitable solvents for the saturatedorganic solution include cyclohexanone, 1-propanol, dimethyl carbonate,N-methylpyrrolidone (NMP), diisopropyl ether, diethyl ether, ethyleneglycol, dimethylformamide (DMF), 2-butanol, cumene, isobutyl acetate,3-methyl-1-butanol, and anisole. Alternatively, the same listed solvents(e.g., cyclohexanone) can be used to form a solution of L-ornithinephenyl acetate, and Form II may be precipitated by adding ethanol atambient conditions. As another example, Form II may also be obtained byforming a slurry of L-ornithine phenyl acetate with the listed organicsolvents and cycling the temperature between 25° and 40° C. every 4hours for about 18 cycles (or 72 hours).

Accordingly, in the context of the processes for making L-ornithinephenyl acetate disclosed above, the process can yield Form II byutilizing particular isolation methods. For example, L-ornithine phenylacetate may by isolated by adding IPA, or evaporating the organicsolvent, to yield Form II.

FIG. 6 shows the crystalline structure of Form II as determined by XRPD.Form II, which may be obtained by the methods disclosed above, exhibitscharacteristic peaks at approximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8°and 24.1° 2θ. Thus, in some embodiments, a crystalline form ofL-ornithine phenyl acetate has one or more characteristic peaks (e.g.,one, two, three, four, five or six characteristic peaks) selected fromapproximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8° and 24.12° θ.

FIG. 7 shows results obtained by differential scanning calorimetry (DSC)for Form II. These results indicate a melting point of about 202° C.,which is approximately the same as the melting point for Form I. Thissuggests that Form I transitions to Form II upon heating above about 35°C. Form II was also analyzed using TG/DTA, as shown in FIG. 8, andexhibits an about 9.7% weight loss associated with residual solvent. Themelting point of about 202° C. could also be observed by TGA testing.Accordingly, in some embodiments, a crystalline form of L-ornithinephenyl acetate exhibits a melting point of about 202° C.

A 7-day stability study of Form II at 40° C./75% RH failed to produce anobservable phase change. In fact, Form II was stable over 14 days whenexposed to elevated temperatures, varying pHs, UV light or oxygen.Accordingly, Form II is considered stable.

FIG. 9 shows nuclear magnetic resonance (NMR) integrals and chemicalshifts for Form II. The integrals confirm the presence of L-ornithinephenyl acetate: 7.5 (aromatic CH), 3.8 (CH adjacent to NH2), 3.6 (CH2unit of phenylacetate), 3.15 (CH2 adjacent to NH2) and 1.9 (aliphaticCH2 units) ppm (integrals: 5:1:2:2:4 protons; 7.0, 1.4, 2.9, 3.0, 5.9).Amine protons and hydroxyl protons were not observed due to protonexchange at both the zwitterion and site of salt formation. Meanwhile,FIG. 10 shows dynamic vapor sorption (DVS) results for Form II, and showa water uptake of about 0.3% by weight. XRPD results following DVAanalysis (not shown) confirm that Form II did not transition to adifferent polymorph. Form II can therefore be characterized asnon-hygroscopic and stable over a wide range of humidity.

Single crystal x-ray diffraction (SXRD) was also used to determine thestructure of Form II at 23° and −123° C., and the results are summarizedin TABLES 3 and 4. The results demonstrate that Form II is anhydrous andtherefore structurally different from Form I. In some embodiments, acrystalline form of L-ornithine phenyl acetate can be represented by theformula C₁₃H₂₀N₂O₄. In some embodiments, a crystalline form ofL-ornithine phenyl acetate can be represented by the formula[C₅H₁₃N₂O₂][C₈H₇O₂]. In some embodiments, a crystalline form ofL-ornithine phenyl acetate exhibits a single crystal X-raycrystallographic analysis with crystal parameters approximately equal tothe following: unit cell dimensions of a=6.594(2) Å, α=90°, b=6.5448(18)Å, β=91.12(3°), c=31.632(8) Å, γ=90°; a monoclinic crystal system; and aP2₁ space group.

TABLE 3 Crystallographic Data of Form II Collected at 23° C. EmpiricalFormula C₁₃H₂₀N₂O₄ or [C₅H₁₃N₂O₂][C₈H₇O₂] Formula Weight 268.31 CrystalSystem Monoclinic Space Group P2₁ Unit Cell Dimensions a = 6.594(2) Å α= 90° b = 6.5448(18) Å β = 91.12(3)° c = 31.632(8) Å γ = 90° Volume1364.9(7) Å³ Number of Reflections 3890 (3° < θ < 20.5°) Density(calculated) 1.306 mg/cm³

TABLE 4 Crystallographic Data of Form II Collected at −123° C. EmpiricalFormula C₁₅ H₂₈ N₂ O₆ or [C₅H₁₃N₂O₂][C₈H₇O₂] Formula Weight 332.39Crystal System Monoclinic Space Group P2₁ Unit Cell Dimensions a =5.3652(4) Å α = 90° b = 7.7136(6) Å β = 94.986(6)° c = 20.9602(18) Å γ =90° Volume 864.16(12) Å³ Number of Reflections 1516 (2.5° < θ < 28°)Density (calculated) 1.277 mg/cm³Form III

The precise conditions for forming crystalline Form III may beempirically determined and it is only possible to give a number ofmethods which have been found to be suitable in practice.

Thus, for example, Form III may be obtained by placing a saturatedsolution of L-ornithine phenyl acetate in a cooled temperatureenvironment of about −21° C., where the solution is a mixture of acetoneand water (e.g., equal parts volume of acetone and water). As anotherexample, adding IPA to a saturated solution of L-ornithine phenylacetate in 2-butanol can yield Form III when completed at ambientconditions. Furthermore, Form III may be obtained, for example, byadding IPA to a saturated solution of L-ornithine phenyl acetate inisobutyl acetate when completed at reduced temperatures of about −21° C.

Accordingly, in the context of the processes for making L-ornithinephenyl acetate disclosed above, the process can yield Form III byutilizing particular solvents and isolation methods. For example,L-ornithine phenyl acetate may be formed within a mixture of acetone andwater, and subsequently placed in a cool environment of about −21° C. toyield Form III.

FIG. 11 shows the crystalline structure of Form III as determined byXRPD. Form III, which may be obtained by the methods disclosed above,exhibits characteristic peaks at approximately 5.8°, 14.1°, 18.6°,19.4°, 22.3° and 24.8° 2θ. Thus, in some embodiments, a crystalline formof L-ornithine phenyl acetate has one or more characteristic peaks(e.g., one, two, three, four, five or six characteristic peaks) selectedfrom approximately 5.8°, 14.1°, 18.6°, 19.4°, 22.3° and 24.8° 2θ.

FIG. 12 shows results obtained by differential scanning calorimetry(DSC) for Form III. These results indicate a melting point of about 203°C., which is approximately the same as the melting points for Form I andForm II. Additionally, Form III exhibits an endotherm at about 40° C.Form III was also analyzed using TG/DTA, as shown in FIG. 13, andexhibits no significant weight loss before the melting point. Form IIImay therefore be characterized as anhydrous. The melting point of about203° C. could also be observed by TGA testing. Accordingly, in someembodiments, a crystalline form of L-ornithine phenyl acetate exhibits amelting point of about 203° C. In some embodiments, a crystalline formof L-ornithine phenyl acetate is characterized by differential scanningcalorimetry as having an endotherm at about 40° C. In some embodiments,a crystalline form of L-ornithine phenyl acetate is anhydrous.

A 7-day stability study of Form III at 40° C./75% RH indicated that atransformation to Form II occurred under these conditions. In contrast,Form II is stable at elevated temperatures, with or without vacuum, forperiods of 7 or 10 days. Accordingly, Form III is most likelymetastable, but more stable than Form I.

FIG. 14 shows nuclear magnetic resonance (NMR) integrals and chemicalshifts for Form III. The integrals confirm the presence of L-ornithinephenyl acetate: 7.5 (aromatic CH), 3.8 (CH adjacent to NH2), 3.6 (CH2unit of phenyl acetate), 3.15 (CH2 adjacent to NH2) and 1.9 (aliphaticCH2 units) ppm (integrals: 5:1:2:2:4 protons; 4.2, 0.8, 1.7, 1.7, 3.0).Amine protons and hydroxyl protons were not observed due to protonexchange at both the zwitterion and site of salt formation. Meanwhile,FIG. 15 shows dynamic vapor sorption (DVS) results for Form III, andshow a water uptake of about 2.0% by weight. XRPD results following DVSanalysis (not shown) confirm that Form III did not transition to adifferent polymorph. Form III therefore exhibits greater water uptakecompared to Forms I and II; however Form III is still characterized asnon-hygroscopic and stable over a wide range of humidity at roomtemperature.

Form V

The precise conditions for forming crystalline Form V may be empiricallydetermined and it is only possible to give a number of methods whichhave been found to be suitable in practice.

Thus, for example, Form V may be obtained by placing a saturatedsolution of L-ornithine phenyl acetate in a cooled temperatureenvironment of about −21° C., where the solution is cyclohexanone. Asanother example, the same saturated solution may yield Form V whenevaporating the solvent.

Form V also forms from saturated solutions of L-ornithine phenyl acetatehaving diisopropyl ether as a solvent. For example, a saturated solutionhaving a solvent ratio of about 1 to 2 of diisopropyl ether and IPA willyield Form V when placed in a cooled temperature environment of about 4°C. Similarly, a solution having only the solvent diisopropyl ether canyield Form V when placed in a cooled temperature environment of about−21° C.

FIG. 16 shows the crystalline structure of Form V as determined by XRPD.

Form V, which may be obtained by the methods disclosed above, exhibitscharacteristic peaks at approximately 13.7°, 17.4°, 19.8°, 20.6° and23.7° 2θ. Thus, in some embodiments, a crystalline form of L-ornithinephenyl acetate has one or more characteristic peaks (e.g., one, two,three, four, or five characteristic peaks) selected from approximately13.7°, 17.4°, 19.8°, 20.6° and 23.7° 2θ.

FIG. 17 shows results obtained by differential scanning calorimetry(DSC) for Form V. These results indicate a melting point of about 196°C., which is below the melting point of other forms. Form V alsoexhibits an endotherm at about 174° C. Form V was also analyzed usingthermal gravimetric analysis (TGA), as shown in FIG. 18, and exhibits nosignificant weight loss before the melting point. Form V may thereforebe characterized as anhydrous. The melting point of about 196° C. couldalso be observed by TGA testing. Accordingly, in some embodiments, acrystalline form of L-ornithine phenyl acetate exhibits a melting pointof about 196° C. In some embodiments, a crystalline form of L-ornithinephenyl acetate is characterized by differential scanning calorimetry ashaving an endotherm at about 174° C. In some embodiments, a crystallineform of L-ornithine phenyl acetate is anhydrous.

FIG. 19 shows nuclear magnetic resonance (NMR) integrals and chemicalshifts for Form V. The integrals confirm the presence of L-ornithinephenyl acetate: 7.5 (aromatic CH), 3.8 (CH adjacent to NH2), 3.6 (CH2unit of phenyl acetate), 3.15 (CH2 adjacent to NH2) and 1.9 (aliphaticCH2 units) ppm (integrals: 5:1:2:2:4 protons; 4.2, 0.8, 1.7, 1.7, 3.0).Amine protons and hydroxyl protons were not observed due to protonexchange at both the zwitterion and site of salt formation. Meanwhile,FIG. 19 shows dynamic vapor sorption (DVS) results for Form V, and showa water uptake of about 0.75% by weight. XRPD results following DVSanalysis (not shown) suggest that Form V transitioned to Form II, butthe chemical composition was unchanged. Form V is thereforecharacterized as non-hygroscopic, but not stable over a wide range ofhumidity.

A 7-day stability study of Form V at 40° C./75% RH indicated that atransformation to Form II occurred under these conditions; however thechemical composition was unchanged. Accordingly, Form V is most likelymetastable.

Methods of Treating Liver Decompensation or Hepatic Encephalopathy

L-Ornithine phenyl acetate, and accordingly any of the compositions ofL-ornithine phenyl acetate disclosed herein, may be administered to asubject for treating or ameliorating the onset of liver decompensationor hepatic encephalopathy. L-Ornithine phenyl acetate can thus beadministered to improve the condition of a subject, for example asubject suffering from chronic liver disease following a precipitatingevent. As another example, L-ornithine phenyl acetate may beadministered to combat or delay the onset of liver decompensation orhepatic encephalopathy.

L-Ornithine phenyl acetate may be administered in combination to asubject for treatment of hepatic encephalopathy. L-Ornithine phenylacetate may be administered to improve the condition of a patientsuffering from hepatic encephalopathy. L-Ornithine phenyl acetate may beadministered to alleviate the symptoms associated with hepaticencephalopathy. L-Ornithine phenyl acetate may be administered to combathepatic encephalopathy. L-Ornithine phenyl acetate may be administeredto prevent or reduce the likelihood of an initial hepaticencephalopathic episode in a person at risk for hepatic encephalopathicepisodes. L-Ornithine phenyl acetate may be administered to lessen theseverity of an initial hepatic encephalopathic episode in a person atrisk for hepatic encephalopathic episodes. L-Ornithine phenyl acetatemay be administered to delay an initial hepatic encephalopathic episodein a person at risk for hepatic encephalopathic episodes.

Development of liver decompensation and hepatic encephalopathy commonlyinvolves “precipitating events” (or “acute attacks”). Such precipitatingevents include gastrointestinal bleeding, infection (sepsis), portalvein thrombosis and dehydration. The onset of such an acute attack islikely to lead to hospitalization. The patient may suffer one of theseacute attacks or a combination of these acute attacks.

A subject who has had or is suspected of having had an acute attack istreated according to the invention with L-ornithine phenyl acetate toprevent or reduce the likelihood of progression of the liver to thedecompensated state. Consequently, L-ornithine phenyl acetate canprevent or reduce the likelihood of the medical consequences of liverdecompensation such as hepatic encephalopathy. L-Ornithine phenylacetate may be used to preserve liver function. Use of L-ornithinephenyl acetate may thus extend the life of a patient with liver disease.In one embodiment, the metabolic consequences of a gastrointestinalbleed such as hyperammonemia, hypoisoleucemia and reduced proteinsynthesis in the post-bleeding period are prevented.

Typically, treatment of subjects may begin as soon as possible after theonset or the suspected onset of a precipitating event (acute attack).Preferably, treatment of the subject begins prior to repeated acuteattacks. More preferably, treatment of the subject begins following thefirst acute attack. Thus, in some embodiments, the subject treated withL-ornithine phenyl acetate is identified as having the onset or thesuspected onset of a precipitating event (acute attack).

Treatment is typically given promptly after the start of an acuteattack. Treatment may begin after the symptom(s) of an acute attack orsuspected acute attack have been detected e.g. by a medic such as aphysician, a paramedic or a nurse. Treatment may begin uponhospitalization of the subject. Treatment may thus begin within 6 hours,within 3 hours, within 2 hours or within 1 hour after the symptom(s) ofan acute attack or suspected acute attack have been detected. Treatmentof the subject may therefore begin from 1 to 48 hours, for example from1 to 36 hours or from 1 to 24 hours after the symptom(s) of an acuteattack or suspected acute attack have been detected.

Treatment may occur for up to 8 weeks, for example up to 6 weeks, up to4 weeks or up to 2 weeks after the symptom(s) of an acute attack orsuspected acute attack have been detected. Treatment may therefore occurfor up to 48 hours, for example for up to 36 hours or for up to 24 hoursafter the symptom(s) of an acute attack or suspected acute attack havebeen detected. Typically, treatment occurs to the time when recoveryfrom the acute precipitating event is evident.

L-Ornithine phenyl acetate may also be used to treat or amelioratehyperammonemia. Thus, L-ornithine phenyl acetate may be administered topatients identified as having excess ammonia levels in the blood, orpatients exhibiting symptoms of excess ammonia in the blood. L-Ornithinephenyl acetate may also be administered to reduce the risk ofhyperammonemia. In some embodiments, L-ornithine phenyl acetate can beadministered daily, for an indefinite period of time. For example, dailydosages may be administered for the life of the patient, or until aphysician determines the patient no longer exhibits a risk forhyperammonemia. In some embodiments, a therapeutically effective amountof L-ornithine phenyl acetate is administered to reduce the risk ofhyperammonemia. In some embodiments, a therapeutically effective amountof L-ornithine phenyl acetate is administered orally for the prophylaxisof hyperammonemia.

A therapeutically effective amount of L-ornithine phenyl acetate isadministered to the subject. As will be readily apparent to one skilledin the art, the useful in vivo dosage to be administered and theparticular mode of administration will vary depending upon the age,weight, the severity of the affliction, and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. (See e.g., Fingl et al. 1975, in “ThePharmacological Basis of Therapeutics”, which is hereby incorporatedherein by reference in its entirety, with particular reference to Ch. 1,p. 1). The determination of effective dosage levels, that is the dosagelevels necessary to achieve the desired result, can be accomplished byone skilled in the art using routine pharmacological methods. Typically,human clinical applications of products are commenced at lower dosagelevels, with dosage level being increased until the desired effect isachieved. Alternatively, acceptable in vitro studies can be used toestablish useful doses and routes of administration of the compositionsidentified by the present methods using established pharmacologicalmethods.

A typical dose of L-ornithine phenyl acetate may be from about 0.02 toabout 1.25 g/kg of bodyweight (preferably from about 0.1 to about 0.6g/kg of bodyweight). A dosage may therefore be from about 500 mg toabout 50 g (preferably about 5 g to about 40 g, and more preferablyabout 10 g to about 30 g).

A single daily dose may be administered. Alternatively, multiple doses,for example two, three, four or five doses may be administered. Suchmultiple doses may be administered over a period of one month or twoweeks or one week. In some embodiments, a single dose or multiple dosessuch as two, three, four or five doses may be administered daily.

EXAMPLES AND EXPERIMENTAL METHODS

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

X-Ray Powder Diffraction (XRPD)

XRPD analysis was carried out on a Bruker D8 advance or Seimens D5000,scanning the samples between 4° and 50° 2θ. In embodiments using theBruker D8 device, approximately 5 mg of a sample was gently compressedon the XRPD zero back ground single 96 well plate sample holder. Thesample was then loaded into a Bruker D8-Discover diffractometer intransmission mode and analysed using the following experimentalconditions.

Operator D8-Discover Raw Data Origin BRUKER-binary V3 (.RAW) Scan AxisGonio Start Position [°2θ.] 4.0000 End Position [°2θ.] 49.9800 Step Size[°2θ.] 0.0200 Scan Step Time [s] 39.1393 Scan Type Continuous Offset[°2θ.] 0.0000 Divergence Slit Type Fixed Divergence Slit Size [°] 2.0000Specimen Length [mm] 10.00 Receiving Slit Size [mm] 0.1000 MeasurementTemperature [° C.] 25.00 Anode Material Cu K-Alpha1 [Å] 1.54060 K-Alpha2[Å] 1.54443 K-Beta [Å] 1.39225 K-A2/K-A1 Ratio 0.50000 GeneratorSettings 40 mA, 40 kV Diffractometer Type Unknown Diffractometer Number0 Goniometer Radius [mm] 250.00 Dist. Focus-Diverg. Slit [mm] 91.00Incident Beam Monochromator No Spinning No

In embodiments using the Seimens D5000 device, approximately 5 mg ofsample was gently compressed on glass slide containing a thin layer ofholding grease. The sample was then loaded into a Seimens D5000diffractometer running in reflection mode and analysed, whilst spinning,using the following experimental conditions.

Raw Data Origin Siemens-binary V2 (.RAW) Start Position [°2θ.] 3.0000End Position [°2θ.] 50.000 Step Size [°2θ.] 0.0200 Scan Step Time [s]0.8 Scan Type Continuous Offset [°2θ.] 0.0000 Divergence Slit Type FixedDivergence Slit Size [°] 1.0000 Specimen Length [mm] various ReceivingSlit Size [mm] 0.2000 Measurement Temperature [° C.] 20.00 AnodeMaterial Cu K-Alpha1 [Å] 1.54060 K-Alpha2 [Å] 1.54443 K-Beta [Å] 1.39225K-A2/K-A1 Ratio 0.50000 (nominal) Generator Settings 40 mA, 40 kVDiffractometer Type d5000 Diffractometer Number 0 Goniometer Radius [mm]217.50 Incident Beam Monochromator No Diffracted Beam Monochromator(Graphite) Spinning YesSingle Crystal X-Ray Diffraction (SXRD)

All measurements were carried out using a Bruker Smart Apexdiffractometer operating with Mo-Kα radiation. Unless otherwisespecified the data were obtained in 60 ω-scan 10 s images collected inthree separate settings of 2θ and φ.

Differential Scanning Calorimetry (DSC)

Approximately 5 mg of sample was weighed into an aluminium DSC pan andsealed with a pierced aluminium lid (non-hermetically). The sample panwas then loaded into a Seiko DSC6200 (equipped with a cooler), cooled,and held at 25° C. Once a stable heat-flow response was obtained, thesample and reference were then heated to about 250° C. at a scan rate of10° C./min and the resulting heat flow response monitored. Prior toanalysis, the instrument was temperature and heat-flow calibrated usingan indium reference standard. Sample analysis was carried out by Musemeasurement software where the temperatures of thermal events werequoted as the onset temperature, measured according to themanufacturer's specifications.

Thermogravimetric Gravimetric/Differential Thermal Analysis (TG/DTA)

Approximately 5 mg of sample was weighed into an aluminium pan andloaded into a simultaneous thermogravimetric/differential thermalanalyser (DTA) and held at room temperature. The sample was then heatedat a rate of 10° C./min from 25° C. to 300° C. during which time thechange in sample weight was monitored along with any thermal events(DTA). Nitrogen was used as the purge gas, at a flow rate of 20 cm³/min.Prior to analysis the instrument was weight and temperature calibratedusing a 100 mg reference weight and an indium reference standard,respectively.

Dynamic Vapor Sorption (DVS)

Approximately 10 mg of sample was placed into a wire-mesh vapor sorptionbalance pan and loaded into a DVS-1 dynamic vapor sorption balancesupplied by Scientific and Medical Systems (SMS). The sample was thendried by maintaining a 0% humidity environment until no further weightchange was recorded. The sample was then subjected to a ramping profilefrom 0-90% relative humidity (RH) at 10% increments, maintaining thesample at each step until a stable weight had been achieved (99.5% stepcompletion). After completion of the sorption cycle, the sample was thendried using the same procedure. The weight change during thesorption/desorption cycles were then plotted, allowing for thehygroscopic nature of the sample to be determined.

¹H Nuclear Magnetic Resonance (NMR)

¹H NMR was performed on a Bruker AC200. An NMR of each sample wasperformed in d-H₂O and each sample was prepared to about 5 mgconcentration. The NMR spectra for L-ornithine benzoate and L-ornithinephenyl acetate are provided in FIGS. 21 and 22, respectively.

Solubility Approximations

Approximately, 25 mg portions of the sample were placed in vials 5volume increments of the appropriate solvent system were added. Betweeneach addition, the mixture was checked for dissolution and if nodissolution was apparent, the mixture was warmed to 50° C., and checkedagain. The procedure was continued until dissolution was observed orwhen 100 volumes of solvent had been added.

HPLC Solubility Determinations

Slurries of each solvent were prepared and the samples shaken for about48 hrs at 25° C. Each sample was then drawn through a filter, and thefiltrate transferred to an HPLC vial for analysis. From the data thesolubility of L-ornithine phenyl acetate for each solvent wasdetermined.

Temperature Cycling Experiments

Using the information gathered from the solubility approximations,slurries of the sample were prepared in 24 selected solvent systems. Theslurries were temperature cycled at 40° C. or 25° C. in 4 hour cyclesfor a period of 72 hours. The solids were visually checked for anyobvious signs of degradation (i.e. color changes) and then, if notdegraded, isolated by filtration. The solids were allowed to dry atambient conditions for about 24 hours prior to analysis.

Crash Cooling Experiments

Crash cooling experiments were performed by placing saturated solutionsof the sample, in the 24 selected solvent systems, in environments of 4°C. and −21° C. for about 48 hours. Any solid material was recovered andthe solids were allowed to dry at ambient conditions for about 24 hoursprior to analysis.

Evaporation Experiments

Evaporation experiments were conducted by allowing saturated solutionsof the sample to evaporate freely at ambient conditions. The solidmaterial was then recovered after the material had evaporated to drynessand analyzed.

Anti-Solvent Addition Experiments

Anti-solvent addition experiments were conducted by adding anti-solventto saturated solutions of the sample. The addition was continued untilthere was no further precipitation and the samples adjusted to varioustemperature for 24 hours: elevated, ambient, 4° C. or −21°. The solidwas then isolated and dried at ambient conditions for about 24 hoursprior to analysis.

Polarized Light Microscopy (PLM)

The presence of crystallinity (birefringence) was determined using aLeica Leitz DMRB polarised optical microscope equipped with a highresolution Leica camera and image capture software (Firecam V.1.0). Allimages were recorded using a 10× objective, unless otherwise stated.

Silver Analysis

All silver analysis was carried out on an Agilent 7500ce ICP-MS.

Intrinsic Dissolution Rates

Approximately 100 mg of each form was compressed into discs by placingthe material into a die (diameter 12 mm) and compressing the die under 5tons of pressure in a hydraulic press for about 2 minutes. Thedissolution instrument, Sotax AT7 conforms to EP2 and USP2 in whichpaddles were used to stir the media. Each form was tested under thefollowing pH conditions; 1.0, 4.5 and 6.7, in the stationary disc mode(i.e. discs were added at time=0 seconds and allowed to sink to thebottom of the media). 1 cm³ aliquots of media were extracted from thedissolution pots at times 10, 20, 30, 40, 50, 60, 70, 80 and 120 secondsand tested for API concentration by HPLC. Dissolution curves wereplotted and from the first 6 or 7 points on the curves the intrinsicdissolution rate curves were calculated. All tests were carried out at37° C. and a paddle speed of 150 rpm.

HPLC-UV Instrument Details

-   -   Instrument: Agilent 1200    -   Column: Gemini C18, 5 μm, 150.0×4.6 mm    -   Column Temperature: 40° C.    -   Mobile Phase A: Phosphate Buffer    -   Mobile Phase B: Acetonitrile    -   Elution: Gradient    -   λ: 210 nm    -   Injection Volume: 10 μL    -   Flow Rate: 1 mL/min        Thin Layer Chromatography (TLC)

A small spot of solution containing the sample was applied to a plate,about one centimeter from the base. The plate is then dipped into theTLC tank (sealed container) containing methanol:ethyl acetate (95:5)solvent mixture. The solvent moves up the plate by capillary action andmeets the sample mixture, which is dissolved and is carried up the plateby the solvent mixture. The number of spots was noted and the Rf valueswere calculated for each spot.

Infrared (IR)

Infrared spectroscopy was carried out on a Bruker ALPHA P spectrometer.Sufficient material was placed onto the centre of the plate of thespectrometer and the spectra were obtained using the followingparameters:

-   -   Resolution: 4 cm-1    -   Background Scan Time: 16 scans    -   Sample Scan Time: 16 scans    -   Data Collection: 4000 to 400 cm-1    -   Result Spectrum: Transmittance    -   Software: OPUS version 6        Stabilities Studies: pH 1, 4, 7, 10 and 14 Environments

Slurries (supersaturated solution: about 250 μl of pH solution and solidwas added until dissolution was no longer observed and ca. 100 mg ofsolid was in the slurry) were prepared for each form in a variety of pHenvironments; 1, 4, 7, 10 and 13.2. The slurries were shaken constantlyfor a period of 14 days and measurements taken at 7 and 14 day timepoints. Appropriate buffers were prepared for each pH and are detailedfurther below.

A buffer having a pH value of 1 was prepared by dissolving 372.75 mg ofpotassium chloride in 25 ml of deionized water to give a 0.2 M solution.Subsequently, 67 ml of 0.2 M hydrochloric acid was added (this wasprepared from a 5 M solution; 10 ml was added to 40 ml of deionizedwater giving a 1 M solution which was diluted further; 20 ml was addedto 80 ml of deionized water giving the required 0.2 M solution) toachieve the desired pH.

A buffer having a pH value of 4 was prepared by dissolving 1.02 g ofpotassium hydrogen phthalate in 50 ml of deionized water to give a 0.1 Msolution.

A buffer having a pH value of 7 was prepared by dissolving 680.00 mg ofpotassium phosphate monobasic in 50 ml of deionized water to give a 0.1M solution. Subsequently, 29.1 ml of 0.1 M sodium hydroxide was added(this was prepared from a 1 M solution; 5 ml was added to 45 ml ofdeionized water giving the required 0.1 M solution) to achieve thedesired pH.

A buffer having a pH value of 10 was prepared by dissolving 210.00 mg ofsodium bicarbonate in 50 ml of deionized water to give a 0.05 Msolution. Subsequently, 10.7 ml of 0.1 M sodium hydroxide was added(this was prepared from a 1 M solution; 5 ml was added to 45 ml ofdeionized water giving the required 0.1 M solution) to achieve thedesired pH.

A buffer having a pH value of pH 13.2 by dissolving 372.75 mg ofpotassium chloride in 25 ml of deionized water to give a 0.2 M solution.Subsequently, 66 ml of 0.2 M sodium hydroxide was added (this wasprepared from a 1 M solution; 20 ml was added to 80 ml of deionizedwater giving the required 0.2 M solution) taking the pH to 13. 1M sodiumhydroxide was then added drop wise to achieve the desired pH.

Example 1: Precipitating Crystalline Forms

Saturated solutions of L-ornithine phenyl acetate were subjected totemperature cycling, crash cooling, evaporation, or anti-solventaddition as described above. The precipitate was analyzed by PLM andXRPD to determine the crystalline form (if any). The results aresummarized in TABLE 5.

Six unique crystalline forms were identified from the precipitationstudies, Forms I-VI. However, Forms IV and VI were obtained fromsolutions of acetic acid, and NMR results confirmed these forms to beL-ornithine acetate. Meanwhile, Tests 540-611 utilized samples ofL-ornithine phenyl acetate originally isolated by the addition ofethanol anti-solvent. Many of these example produced Form I, which is anethanol solvate, and therefore it is believed these samples originallyincluded residual ethanol. Consequently, Form I may not be reproducedfor certain conditions if the original sample does not include residualethanol.

TABLE 5 Examples of Preparing Crystalline Forms Crystallization TestMethod Solvent Results 1 Temp. Cycling cyclohexanone Form II 2Controlled Cool (4° C.) cyclohexanone No Solid 3 Controlled Cool (−21°C.) cyclohexanone Form V 4 Evaporation cyclohexanone Form V 5Anti-Solvent (IPA) cyclohexanone No Solid Addition Elevated Temperature6 Anti-Solvent (IPA) cyclohexanone Form II Addition Ambient Temperature7 Anti-Solvent (IPA) cyclohexanone Form II Addition (4° C.) 8Anti-Solvent (IPA) cyclohexanone Form II Addition (−21° C.) 9Anti-Solvent (Ethanol) cyclohexanone Form II Addition AmbientTemperature 10 Anti-Solvent (Ethanol) cyclohexanone Form I Addition (4°C.) 11 Anti-Solvent (Ethanol) cyclohexanone Form I Addition (−21° C.) 12Temp. Cycling ethanol/acetone Form II (50:50) 13 Controlled Cool (4° C.)ethanol/acetone No Solid (50:50) 14 Controlled Cool (−21° C.)ethanol/acetone Form III (50:50) 15 Evaporation ethanol/acetone Form II(50:50) 16 Anti-Solvent (IPA) ethanol/acetone Form II Addition Elevated(50:50) Temperature 17 Anti-Solvent (IPA) ethanol/acetone Form IIAddition Ambient (50:50) Temperature 18 Anti-Solvent (IPA)ethanol/acetone Form II Addition (4° C.) (50:50) 19 Anti-Solvent (IPA)ethanol/acetone Form II Addition (−21° C.) (50:50) 20 Anti-Solvent(Ethanol) ethanol/acetone Form II Addition Ambient (50:50) Temperature21 Anti-Solvent (Ethanol) ethanol/acetone Form I Addition (4° C.)(50:50) 22 Anti-Solvent (Ethanol) ethanol/acetone Form I Addition (−21°C.) (50:50) 23 Temp. Cycling acetic acid Form IV 24 Controlled Cool (4°C.) acetic acid No Solid 25 Controlled Cool (−21° C.) acetic acid NoSolid 26 Evaporation acetic acid Form II 27 Anti-Solvent (IPA) aceticacid Form VI Addition Elevated Temperature 28 Anti-Solvent (IPA) aceticacid Form IV Addition Ambient Temperature 29 Anti-Solvent (IPA) aceticacid Form IV Addition (4° C.) 30 Anti-Solvent (IPA) acetic acid Form IVAddition (−21° C.) 31 Anti-Solvent (Ethanol) acetic acid Form IVAddition Ambient Temperature 32 Anti-Solvent (Ethanol) acetic acid FormIV Addition (4° C.) 33 Anti-Solvent (Ethanol) acetic acid Form IVAddition (−21° C.) 34 Temp. Cycling 1-propanol Form II 35 ControlledCool (4° C.) 1-propanol Form II 36 Controlled Cool (−21° C.) 1-propanolForm II 37 Evaporation 1-propanol Form II 38 Anti-Solvent (IPA)1-propanol Form II Addition Elevated Temperature 39 Anti-Solvent (IPA)1-propanol Form II Addition Ambient Temperature 40 Anti-Solvent (IPA)1-propanol Form II Addition (4° C.) 41 Anti-Solvent (IPA) 1-propanolForm II Addition (−21° C.) 42 Anti-Solvent (Ethanol) 1-propanol Form IIAddition Ambient Temperature 43 Anti-Solvent (Ethanol) 1-propanol FormI/II Addition (4° C.) 44 Anti-Solvent (Ethanol) 1-propanol Form IAddition (−21° C.) 45 Temp. Cycling dimethylcarbonate Form II 46Controlled Cool (4° C.) dimethylcarbonate No Solid 47 Controlled Cool(−21° C.) dimethylcarbonate Form II 48 Evaporation dimethylcarbonateForm II 49 Anti-Solvent (IPA) dimethylcarbonate Form II AdditionElevated Temperature 50 Anti-Solvent (IPA) dimethylcarbonate Form IIAddition Ambient Temperature 51 Anti-Solvent (IPA) dimethylcarbonateForm II Addition (4° C.) 52 Anti-Solvent (IPA) dimethylcarbonate Form IIAddition (−21° C.) 53 Anti-Solvent (Ethanol) dimethylcarbonate Form IIAddition Ambient Temperature 54 Anti-Solvent (Ethanol) dimethylcarbonateForm I Addition (4° C.) 55 Anti-Solvent (Ethanol) dimethylcarbonate FormII Addition (−21° C.) 56 Temp. Cycling NMP Form II 57 Controlled Cool(4° C.) NMP Form II 58 Controlled Cool (−21° C.) NMP Form II 59Evaporation NMP Form II 60 Anti-Solvent (IPA) NMP Form II AdditionElevated Temperature 61 Anti-Solvent (IPA) NMP Form II Addition AmbientTemperature 62 Anti-Solvent (IPA) NMP Form II Addition (4° C.) 63Anti-Solvent (IPA) NMP Form II Addition (−21° C.) 64 Anti-Solvent(Ethanol) NMP Form II Addition Ambient Temperature 65 Anti-Solvent(Ethanol) NMP Form I/II Addition (4° C.) 66 Anti-Solvent (Ethanol) NMPForm II Addition (−21° C.) 67 Temp. Cycling EtOAc/cyclohexane Form II(1:2) 68 Controlled Cool (4° C.) EtOAc/cyclohexane No Solid (1:2) 69Controlled Cool (−21° C.) EtOAc/cyclohexane No Solid (1:2) 70Evaporation etOAc/cyclohexane Form II (1:2) 71 Anti-Solvent (IPA)etOAc/cyclohexane Form II Addition Elevated (1:2) Temperature 72Anti-Solvent (IPA) etOAc/cyclohexane Form II Addition Ambient (1:2)Temperature 73 Anti-Solvent (IPA) etOAc/cyclohexane Form II Addition (4°C.) (1:2) 74 Anti-Solvent (IPA) etOAc/cyclohexane Form II Addition (−21°C.) (1:2) 75 Anti-Solvent (Ethanol) etOAc/cyclohexane Form II AdditionAmbient (1:2) Temperature 76 Anti-Solvent (Ethanol) etOAc/cyclohexaneForm I Addition (4° C.) (1:2) 77 Anti-Solvent (Ethanol)etOAc/cyclohexane Form I/II Addition (−21° C.) (1:2) 78 Temp. CyclingetOAc/toluene (1:2) Form II 79 Controlled Cool (4° C.) etOAc/toluene(1:2) No Solid 80 Controlled Cool (−21° C.) etOAc/toluene (1:2) Form II81 Evaporation etOAc/toluene (1:2) Form II 82 Anti-Solvent (IPA)etOAc/toluene (1:2) Form II Addition Elevated Temperature 83Anti-Solvent (IPA) etOAc/toluene (1:2) Form II Addition AmbientTemperature 84 Anti-Solvent (IPA) etOAc/toluene (1:2) Form II Addition(4° C.) 85 Anti-Solvent (IPA) etOAc/toluene (1:2) Form II Addition (−21°C.) 86 Anti-Solvent (Ethanol) etOAc/toluene (1:2) Form II AdditionAmbient Temperature 87 Anti-Solvent (Ethanol) etOAc/toluene (1:2) Form IAddition (4° C.) 88 Anti-Solvent (Ethanol) etOAc/toluene (1:2) Form IIAddition (−21° C.) 89 Temp. Cycling IPA/diisopropyl ether Form II (1:2)90 Controlled Cool (4° C.) IPA/diisopropyl ether Form V (1:2) 91Controlled Cool (−21° C.) IPA/diisopropyl ether Form II (1:2) 92Evaporation IPA/diisopropyl ether Form II (1:2) 93 Anti-Solvent (IPA)IPA/diisopropyl ether Form II Addition Elevated (1:2) Temperature 94Anti-Solvent (IPA) IPA/diisopropyl ether Form II Addition Ambient (1:2)Temperature 95 Anti-Solvent (IPA) IPA/diisopropyl ether Form II Addition(4° C.) (1:2) 96 Anti-Solvent (IPA) IPA/diisopropyl ether Form IIAddition (−21° C.) (1:2) 97 Anti-Solvent (Ethanol) IPA/diisopropyl etherForm II Addition Ambient (1:2) Temperature 98 Anti-Solvent (Ethanol)IPA/diisopropyl ether Form I Addition (4° C.) (1:2) 99 Anti-Solvent(Ethanol) IPA/diisopropyl ether Form I/II Addition (−21° C.) (1:2) 100Temp. Cycling DIPE Form II 101 Controlled Cool (4° C.) DIPE No Solid 102Controlled Cool (−21° C.) DIPE Form V 103 Evaporation DIPE Form II 104Anti-Solvent (IPA) DIPE Form II Addition Elevated Temperature 105Anti-Solvent (IPA) DIPE Form II Addition Ambient Temperature 106Anti-Solvent (IPA) DIPE Form II Addition (4° C.) 107 Anti-Solvent (IPA)DIPE Form II Addition (−21° C.) 108 Anti-Solvent (Ethanol) DIPE Form IIAddition Ambient Temperature 109 Anti-Solvent (Ethanol) DIPE Form IAddition (4° C.) 110 Anti-Solvent (Ethanol) DIPE Form II Addition (−21°C.) 111 Temp. Cycling nitromethane/water No Solid (20%) 112 ControlledCool (4° C.) nitromethane/water No Solid (20%) 113 Controlled Cool (−21°C.) nitromethane/water No Solid (20%) 114 Evaporation nitromethane/waterForm II (20%) 115 Anti-Solvent (IPA) nitromethane/water No SolidAddition Elevated (20%) Temperature 116 Anti-Solvent (IPA)nitromethane/water Form II Addition Ambient (20%) Temperature 117Anti-Solvent (IPA) nitromethane/water Form II Addition (4° C.) (20%) 118Anti-Solvent (IPA) nitromethane/water Form II Addition (−21° C.) (20%)119 Anti-Solvent (Ethanol) nitromethane/water Form II Addition Ambient(20%) Temperature 120 Anti-Solvent (Ethanol) nitromethane/water Form IAddition (4° C.) (20%) 121 Anti-Solvent (Ethanol) nitromethane/waterForm I/II Addition (−21° C.) (20%) 122 Temp. Cycling acetone/water (20%)No Solid 123 Controlled Cool (4° C.) acetone/water (20%) Form II 124Controlled Cool (−21° C.) acetone/water (20%) Form II 125 Evaporationacetone/water (20%) Form II 126 Anti-Solvent (IPA) acetone/water (20%)Form II Addition Elevated Temperature 127 Anti-Solvent (IPA)acetone/water (20%) Form II Addition Ambient Temperature 128Anti-Solvent (IPA) acetone/water (20%) Form II Addition (4° C.) 129Anti-Solvent (IPA) acetone/water (20%) Form II Addition (−21° C.) 130Anti-Solvent (Ethanol) acetone/water (20%) Form II Addition AmbientTemperature 131 Anti-Solvent (Ethanol) acetone/water (20%) Form IAddition (4° C.) 132 Anti-Solvent (Ethanol) acetone/water (20%) Form IIAddition (−21° C.) 133 Temp. Cycling 1,4 dioxane/water Form II (20%) 134Controlled Cool (4° C.) 1,4 dioxane/water Form II (20%) 135 ControlledCool (−21° C.) 1,4 dioxane/water No Solid (20%) 136 Evaporation 1,4dioxane/water Form II (20%) 137 Anti-Solvent (IPA) 1,4 dioxane/waterForm II Addition Elevated (20%) Temperature 138 Anti-Solvent (IPA) 1,4dioxane/water Form II Addition Ambient (20%) Temperature 139Anti-Solvent (IPA) 1,4 dioxane/water Form II Addition (4° C.) (20%) 140Anti-Solvent (IPA) 1,4 dioxane/water Form II Addition (−21° C.) (20%)141 Anti-Solvent (Ethanol) 1,4 dioxane/water Form II Addition Ambient(20%) Temperature 142 Anti-Solvent (Ethanol) 1,4 dioxane/water Form IAddition (4° C.) (20%) 143 Anti-Solvent (Ethanol) 1,4 dioxane/water FormII Addition (−21° C.) (20%) 144 Temp. Cycling diethyl ether Form II 145Controlled Cool (4° C.) diethyl ether No Solid 146 Controlled Cool (−21°C.) diethyl ether No Solid 147 Evaporation diethyl ether Form II 148Anti-Solvent (IPA) diethyl ether Form II Addition Elevated Temperature149 Anti-Solvent (IPA) diethyl ether Form II Addition AmbientTemperature 150 Anti-Solvent (IPA) diethyl ether Form II Addition (4°C.) 151 Anti-Solvent (IPA) diethyl ether Form II Addition (−21° C.) 152Anti-Solvent (Ethanol) diethyl ether Form II Addition AmbientTemperature 153 Anti-Solvent (Ethanol) diethyl ether Form I Addition (4°C.) 154 Anti-Solvent (Ethanol) diethyl ether Form I/II Addition (−21°C.) 155 Temp. Cycling ethylene glycol Form II 156 Controlled Cool (4°C.) ethylene glycol No Solid 157 Controlled Cool (−21° C.) ethyleneglycol No Solid 158 Evaporation ethylene glycol No Solid 159Anti-Solvent (IPA) ethylene glycol Form II Addition Elevated Temperature160 Anti-Solvent (IPA) ethylene glycol Form II Addition AmbientTemperature 161 Anti-Solvent (IPA) ethylene glycol Form II Addition (4°C.) 162 Anti-Solvent (IPA) ethylene glycol Form II Addition (−21° C.)163 Anti-Solvent (Ethanol) ethylene glycol Form II Addition AmbientTemperature 164 Anti-Solvent (Ethanol) ethylene glycol Form II Addition(4° C.) 165 Anti-Solvent (Ethanol) ethylene glycol Form II Addition(−21° C.) 166 Temp. Cycling meOAc/water (20%) No Solid 167 ControlledCool (4° C.) meOAc/water (20%) No Solid 168 Controlled Cool (−21° C.)meOAc/water (20%) No Solid 169 Evaporation meOAc/water (20%) Form II 170Anti-Solvent (IPA) meOAc/water (20%) Form II Addition ElevatedTemperature 171 Anti-Solvent (IPA) meOAc/water (20%) Form II AdditionAmbient Temperature 172 Anti-Solvent (IPA) meOAc/water (20%) Form IIAddition (4° C.) 173 Anti-Solvent (IPA) meOAc/water (20%) Form IIAddition (−21° C.) 174 Anti-Solvent (Ethanol) meOAc/water (20%) Form IIAddition Ambient Temperature 175 Anti-Solvent (Ethanol) meOAc/water(20%) Form I/II Addition (4° C.) 176 Anti-Solvent (Ethanol) meOAc/water(20%) Form II Addition (−21° C.) 177 Temp. Cycling meOH/acetone Form II(50:50) 178 Controlled Cool (4° C.) meOH/acetone No Solid (50:50) 179Controlled Cool (−21° C.) meOH/acetone No Solid (50:50) 180 EvaporationmeOH/acetone Form II (50:50) 181 Anti-Solvent (IPA) meOH/acetone Form IIAddition Elevated (50:50) Temperature 182 Anti-Solvent (IPA)meOH/acetone Form II Addition Ambient (50:50) Temperature 183Anti-Solvent (IPA) meOH/acetone Form II Addition (4° C.) (50:50) 184Anti-Solvent (IPA) meOH/acetone Form II Addition (−21° C.) (50:50) 185Anti-Solvent (Ethanol) meOH/acetone Form II Addition Ambient (50:50)Temperature 186 Anti-Solvent (Ethanol) meOH/acetone Form I Addition (4°C.) (50:50) 187 Anti-Solvent (Ethanol) meOH/acetone Form I/II Addition(−21° C.) (50:50) 188 Temp. Cycling DMF Form II 189 Controlled Cool (4°C.) DMF Form II 190 Controlled Cool (−21° C.) DMF Form II 191Evaporation DMF Form II 192 Anti-Solvent (IPA) DMF Form II AdditionElevated Temperature 193 Anti-Solvent (IPA) DMF Form II Addition AmbientTemperature 194 Anti-Solvent (IPA) DMF Form II Addition (4° C.) 195Anti-Solvent (IPA) DMF Form II Addition (−21° C.) 196 Anti-Solvent(Ethanol) DMF Form II Addition Ambient Temperature 197 Anti-Solvent(Ethanol) DMF Form I/II Addition (4° C.) 198 Anti-Solvent (Ethanol) DMFForm II Addition (−21° C.) 199 Temp. Cycling 2-butanol Form II 200Controlled Cool (4° C.) 2-butanol No Solid 201 Controlled Cool (−21° C.)2-butanol No Solid 202 Evaporation 2-butanol Form II 203 Anti-Solvent(IPA) 2-butanol Form III Addition Elevated Temperature 204 Anti-Solvent(IPA) 2-butanol Form II Addition Ambient Temperature 205 Anti-Solvent(IPA) 2-butanol Form II Addition (4° C.) 206 Anti-Solvent (IPA)2-butanol Form II Addition (−21° C.) 207 Anti-Solvent (Ethanol)2-butanol Form II Addition Ambient Temperature 208 Anti-Solvent(Ethanol) 2-butanol Form I/II Addition (4° C.) 209 Anti-Solvent(Ethanol) 2-butanol Form I/II Addition (−21° C.) 210 Temp. Cyclingcumene Form II 211 Controlled Cool (4° C.) cumene No Solid 212Controlled Cool (−21° C.) cumene No Solid 213 Evaporation cumene Form II214 Anti-Solvent (IPA) cumene Form II Addition Elevated Temperature 215Anti-Solvent (IPA) cumene Form II Addition Ambient Temperature 216Anti-Solvent (IPA) cumene Form II Addition (4° C.) 217 Anti-Solvent(IPA) cumene Form II Addition (−21° C.) 218 Anti-Solvent (Ethanol)cumene Form II Addition Ambient Temperature 219 Anti-Solvent (Ethanol)cumene Form II Addition (4° C.) 220 Anti-Solvent (Ethanol) cumene FormI/II Addition (−21° C.) 221 Temp. Cycling ethyl formate Form II 222Controlled Cool (4° C.) ethyl formate No Solid 223 Controlled Cool (−21°C.) ethyl formate Form II 224 Evaporation ethyl formate Form II 225Anti-Solvent (IPA) ethyl formate Form II Addition Elevated Temperature226 Anti-Solvent (IPA) ethyl formate Form II Addition AmbientTemperature 227 Anti-Solvent (IPA) ethyl formate Form II Addition (4°C.) 228 Anti-Solvent (IPA) ethyl formate Form II Addition (−21° C.) 229Anti-Solvent (Ethanol) ethyl formate Form II Addition AmbientTemperature 230 Anti-Solvent (Ethanol) ethyl formate Form I Addition (4°C.) 231 Anti-Solvent (Ethanol) ethyl formate Form I/II Addition (−21°C.) 232 Temp. Cycling isobutyl acetate Form II 233 Controlled Cool (4°C.) isobutyl acetate No Solid 234 Controlled Cool (−21° C.) isobutylacetate Form II 235 Evaporation isobutyl acetate No Solid 236Anti-Solvent (IPA) isobutyl acetate Form II Addition ElevatedTemperature 237 Anti-Solvent (IPA) isobutyl acetate Form II AdditionAmbient Temperature 238 Anti-Solvent (IPA) isobutyl acetate Form IIAddition (4° C.) 239 Anti-Solvent (IPA) isobutyl acetate Form IIIAddition (−21° C.) 240 Anti-Solvent (Ethanol) isobutyl acetate Form IIAddition Ambient Temperature 241 Anti-Solvent (Ethanol) isobutyl acetateForm II Addition (4° C.) 242 Anti-Solvent (Ethanol) isobutyl acetateForm I/II Addition (−21° C.) 243 Temp. Cycling 3-methyl-1-butanol FormII 244 Controlled Cool (4° C.) 3-methyl-1-butanol No Solid 245Controlled Cool (−21° C.) 3-methyl-1-butanol No Solid 246 Evaporation3-methyl-1-butanol Form II 247 Anti-Solvent (IPA) 3-methyl-1-butanolForm II Addition Elevated Temperature 248 Anti-Solvent (IPA)3-methyl-1-butanol Form II Addition Ambient Temperature 249 Anti-Solvent(IPA) 3-methyl-1-butanol Form II Addition (4° C.) 250 Anti-Solvent (IPA)3-methyl-1-butanol Form II Addition (−21° C.) 251 Anti-Solvent (Ethanol)3-methyl-1-butanol Form II Addition Ambient Temperature 252 Anti-Solvent(Ethanol) 3-methyl-1-butanol Form I/II Addition (4° C.) 253 Anti-Solvent(Ethanol) 3-methyl-1-butanol Form I/II Addition (−21° C.) 254 Temp.Cycling anisole Form II 255 Controlled Cool (4° C.) anisole No Solid 256Controlled Cool (−21° C.) anisole Form II 257 Evaporation anisole FormII 258 Anti-Solvent (IPA) anisole Form II Addition Elevated Temperature259 Anti-Solvent (IPA) anisole Form II Addition Ambient Temperature 260Anti-Solvent (IPA) anisole Form II Addition (4° C.) 261 Anti-Solvent(IPA) anisole Form II/IV Addition (−21° C.) 262 Anti-Solvent (Ethanol)anisole Form II Addition Ambient Temperature 263 Anti-Solvent (Ethanol)anisole Form II Addition (4° C.) 264 Anti-Solvent (Ethanol) anisole FormI Addition (−21° C.) 265 Temp. Cycling IPA/isopropyl acetate Form II(1:2) 266 Controlled Cool (4° C.) IPA/isopropyl acetate No Solid (1:2)267 Controlled Cool (−21° C.) IPA/isopropyl acetate No Solid (1:2) 268Evaporation IPA/isopropyl acetate Form II (1:2) 269 Anti-Solvent (IPA)IPA/isopropyl acetate Form II Addition Elevated (1:2) Temperature 270Anti-Solvent (IPA) IPA/isopropyl acetate Form II Addition Ambient (1:2)Temperature 271 Anti-Solvent (IPA) IPA/isopropyl acetate Form IIAddition (4° C.) (1:2) 272 Anti-Solvent (IPA) IPA/isopropyl acetate FormII Addition (−21° C.) (1:2) 273 Anti-Solvent (Ethanol) IPA/isopropylacetate Form II Addition Ambient (1:2) Temperature 274 Anti-Solvent(Ethanol) IPA/isopropyl acetate Form II Addition (4° C.) (1:2) 275Anti-Solvent (Ethanol) IPA/isopropyl acetate Form I Addition (−21° C.)(1:2) 276 Temp. Cycling EtOH: 1% H₂0 Form II 277 Controlled Cool (4° C.)EtOH: 1% H₂0 No Solid 278 Controlled Cool (−21° C.) EtOH: 1% H₂0 NoSolid 279 Evaporation EtOH: 1% H₂0 No Solid 280 Anti-Solvent (IPA) EtOH:1% H₂0 No Solid Addition Elevated Temperature 281 Anti-Solvent (IPA)EtOH: 1% H₂0 No Solid Addition Ambient Temperature 282 Anti-Solvent(IPA) EtOH: 1% H₂0 No Solid Addition (4° C.) 283 Anti-Solvent (IPA)EtOH: 1% H₂0 No Solid Addition (−21° C.) 284 Anti-Solvent (Ethanol)EtOH: 1% H₂0 No Solid Addition Ambient Temperature 285 Anti-Solvent(Ethanol) EtOH: 1% H₂0 No Solid Addition (4° C.) 286 Anti-Solvent(Ethanol) EtOH: 1% H₂0 No Solid Addition (−21° C.) 287 Temp. CyclingEtOH: 3% H₂0 Form II 288 Controlled Cool (4° C.) EtOH: 3% H₂0 No Solid289 Controlled Cool (−21° C.) EtOH: 3% H₂0 No Solid 290 EvaporationEtOH: 3% H₂0 No Solid 291 Anti-Solvent (IPA) EtOH: 3% H₂0 No SolidAddition Elevated Temperature 292 Anti-Solvent (IPA) EtOH: 3% H₂0 NoSolid Addition Ambient Temperature 293 Anti-Solvent (IPA) EtOH: 3% H₂0No Solid Addition (4° C.) 294 Anti-Solvent (IPA) EtOH: 3% H₂0 No SolidAddition (−21° C.) 295 Anti-Solvent (Ethanol) EtOH: 3% H₂0 No SolidAddition Ambient Temperature 296 Anti-Solvent (Ethanol) EtOH: 3% H₂0 NoSolid Addition (4° C.) 297 Anti-Solvent (Ethanol) EtOH: 3% H₂0 No SolidAddition (−21° C.) 298 Temp. Cycling EtOH: 5% H₂0 Form II 299 ControlledCool (4° C.) EtOH: 5% H₂0 No Solid 300 Controlled Cool (−21° C.) EtOH:5% H₂0 No Solid 301 Evaporation EtOH: 5% H₂0 Form II 302 Anti-Solvent(IPA) EtOH: 5% H₂0 No Solid Addition Elevated Temperature 303Anti-Solvent (IPA) EtOH: 5% H₂0 Form II Addition Ambient Temperature 304Anti-Solvent (IPA) EtOH: 5% H₂0 No Solid Addition (4° C.) 305Anti-Solvent (IPA) EtOH: 5% H₂0 No Solid Addition (−21° C.) 306Anti-Solvent (Ethanol) EtOH: 5% H₂0 No Solid Addition AmbientTemperature 307 Anti-Solvent (Ethanol) EtOH: 5% H₂0 No Solid Addition(4° C.) 308 Anti-Solvent (Ethanol) EtOH: 5% H₂0 No Solid Addition (−21°C.) 309 Temp. Cycling IPA: 1% H₂0 Form II 310 Controlled Cool (4° C.)IPA: 1% H₂0 No Solid 311 Controlled Cool (−21° C.) IPA: 1% H₂0 No Solid312 Evaporation IPA: 1% H₂0 No Solid 313 Anti-Solvent (IPA) IPA: 1% H₂0No Solid Addition Elevated Temperature 314 Anti-Solvent (IPA) IPA: 1%H₂0 No Solid Addition Ambient Temperature 315 Anti-Solvent (IPA) IPA: 1%H₂0 No Solid Addition (4° C.) 316 Anti-Solvent (IPA) IPA: 1% H₂0 NoSolid Addition (−21° C.) 317 Anti-Solvent (Ethanol) IPA: 1% H₂0 No SolidAddition Ambient Temperature 318 Anti-Solvent (Ethanol) IPA: 1% H₂0 NoSolid Addition (4° C.) 319 Anti-Solvent (Ethanol) IPA: 1% H₂0 No SolidAddition (−21° C.) 320 Temp. Cycling IPA: 3% H₂0 Form II 321 ControlledCool (4° C.) IPA: 3% H₂0 No Solid 322 Controlled Cool (−21° C.) IPA: 3%H₂0 No Solid 323 Evaporation IPA: 3% H₂0 No Solid 324 Anti-Solvent (IPA)IPA: 3% H₂0 No Solid Addition Elevated Temperature 325 Anti-Solvent(IPA) IPA: 3% H₂0 No Solid Addition Ambient Temperature 326 Anti-Solvent(IPA) IPA: 3% H₂0 No Solid Addition (4° C.) 327 Anti-Solvent (IPA) IPA:3% H₂0 No Solid Addition (−21° C.) 328 Anti-Solvent (Ethanol) IPA: 3%H₂0 No Solid Addition Ambient Temperature 329 Anti-Solvent (Ethanol)IPA: 3% H₂0 No Solid Addition (4° C.) 330 Anti-Solvent (Ethanol) IPA: 3%H₂0 No Solid Addition (−21° C.) 331 Temp. Cycling IPA: 5% H₂0 Form II332 Controlled Cool (4° C.) IPA: 5% H₂0 No Solid 333 Controlled Cool(−21° C.) IPA: 5% H₂0 No Solid 334 Evaporation IPA: 5% H₂0 Form II 335Anti-Solvent (IPA) IPA: 5% H₂0 No Solid Addition Elevated Temperature336 Anti-Solvent (IPA) IPA: 5% H₂0 No Solid Addition Ambient Temperature337 Anti-Solvent (IPA) IPA: 5% H₂0 No Solid Addition (4° C.) 338Anti-Solvent (IPA) IPA: 5% H₂0 No Solid Addition (−21° C.) 339Anti-Solvent (Ethanol) IPA: 5% H₂0 No Solid Addition Ambient Temperature340 Anti-Solvent (Ethanol) IPA: 5% H₂0 No Solid Addition (4° C.) 341Anti-Solvent (Ethanol) IPA: 5% H₂0 No Solid Addition (−21° C.) 342 Temp.Cycling ACN: 1% H₂0 Form II 343 Controlled Cool (4° C.) ACN: 1% H₂0 NoSolid 344 Controlled Cool (−21° C.) ACN: 1% H₂0 No Solid 345 EvaporationACN: 1% H₂0 Form II 346 Anti-Solvent (IPA) ACN: 1% H₂0 No Solid AdditionElevated Temperature 347 Anti-Solvent (IPA) ACN: 1% H₂0 No SolidAddition Ambient Temperature 348 Anti-Solvent (IPA) ACN: 1% H₂0 No SolidAddition (4° C.) 349 Anti-Solvent (IPA) ACN: 1% H₂0 No Solid Addition(−21° C.) 350 Anti-Solvent (Ethanol) ACN: 1% H₂0 No Solid AdditionAmbient Temperature 351 Anti-Solvent (Ethanol) ACN: 1% H₂0 No SolidAddition (4° C.) 352 Anti-Solvent (Ethanol) ACN: 1% H₂0 No SolidAddition (−21° C.) 353 Temp. Cycling ACN: 6% H₂0 Form II 354 ControlledCool (4° C.) ACN: 6% H₂0 No Solid 355 Controlled Cool (−21° C.) ACN: 6%H₂0 No Solid 356 Evaporation ACN: 6% H₂0 Form II 357 Anti-Solvent (IPA)ACN: 6% H₂0 No Solid Addition Elevated Temperature 358 Anti-Solvent(IPA) ACN: 6% H₂0 No Solid Addition Ambient Temperature 359 Anti-Solvent(IPA) ACN: 6% H₂0 No Solid Addition (4° C.) 360 Anti-Solvent (IPA) ACN:6% H₂0 No Solid Addition (−21° C.) 361 Anti-Solvent (Ethanol) ACN: 6%H₂0 No Solid Addition Ambient Temperature 362 Anti-Solvent (Ethanol)ACN: 6% H₂0 No Solid Addition (4° C.) 363 Anti-Solvent (Ethanol) ACN: 6%H₂0 No Solid Addition (−21° C.) 364 Temp. Cycling ACN: 12% H₂0 No Solid365 Controlled Cool (4° C.) ACN: 12% H₂0 No Solid 366 Controlled Cool(−21° C.) ACN: 12% H₂0 No Solid 367 Evaporation ACN: 12% H₂0 Form II 368Anti-Solvent (IPA) ACN: 12% H₂0 No Solid Addition Elevated Temperature369 Anti-Solvent (IPA) ACN: 12% H₂0 Form II Addition Ambient Temperature370 Anti-Solvent (IPA) ACN: 12% H₂0 No Solid Addition (4° C.) 371Anti-Solvent (IPA) ACN: 12% H₂0 Form II Addition (−21° C.) 372Anti-Solvent (Ethanol) ACN: 12% H₂0 No Solid Addition AmbientTemperature 373 Anti-Solvent (Ethanol) ACN: 12% H₂0 No Solid Addition(4° C.) 374 Anti-Solvent (Ethanol) ACN: 12% H₂0 No Solid Addition (−21°C.) 375 Temp. Cycling DMF: 5% H₂0 Form II 376 Controlled Cool (4° C.)DMF: 5% H₂0 No Solid 377 Controlled Cool (−21° C.) DMF: 5% H₂0 No Solid378 Evaporation DMF: 5% H₂0 No Solid 379 Anti-Solvent (IPA) DMF: 5% H₂0No Solid Addition Elevated Temperature 380 Anti-Solvent (IPA) DMF: 5%H₂0 No Solid Addition Ambient Temperature 381 Anti-Solvent (IPA) DMF: 5%H₂0 No Solid Addition (4° C.) 382 Anti-Solvent (IPA) DMF: 5% H₂0 NoSolid Addition (−21° C.) 383 Anti-Solvent (Ethanol) DMF: 5% H₂0 No SolidAddition Ambient Temperature 384 Anti-Solvent (Ethanol) DMF: 5% H₂0 NoSolid Addition (4° C.) 385 Anti-Solvent (Ethanol) DMF: 5% H₂0 No SolidAddition (−21° C.) 386 Temp. Cycling DMF: 15% H₂0 Form II 387 ControlledCool (4° C.) DMF: 15% H₂0 No Solid 388 Controlled Cool (−21° C.) DMF:15% H₂0 No Solid 389 Evaporation DMF: 15% H₂0 No Solid 390 Anti-Solvent(IPA) DMF: 15% H₂0 No Solid Addition Elevated Temperature 391Anti-Solvent (IPA) DMF: 15% H₂0 No Solid Addition Ambient Temperature392 Anti-Solvent (IPA) DMF: 15% H₂0 No Solid Addition (4° C.) 393Anti-Solvent (IPA) DMF: 15% H₂0 No Solid Addition (−21° C.) 394Anti-Solvent (Ethanol) DMF: 15% H₂0 No Solid Addition AmbientTemperature 395 Anti-Solvent (Ethanol) DMF: 15% H₂0 No Solid Addition(4° C.) 396 Anti-Solvent (Ethanol) DMF: 15% H₂0 No Solid Addition (−21°C.) 397 Temp. Cycling DMF: 30% H₂0 Form II 398 Controlled Cool (4° C.)DMF: 30% H₂0 Form II 399 Controlled Cool (−21° C.) DMF: 30% H₂0 Form II400 Evaporation DMF: 30% H₂0 Form II 401 Anti-Solvent (IPA) DMF: 30% H₂0Form II Addition Elevated Temperature 402 Anti-Solvent (IPA) DMF: 30%H₂0 Form II Addition Ambient Temperature 403 Anti-Solvent (IPA) DMF: 30%H₂0 Form II Addition (4° C.) 404 Anti-Solvent (IPA) DMF: 30% H₂0 Form IIAddition (−21° C.) 405 Anti-Solvent (Ethanol) DMF: 30% H₂0 Form IIAddition Ambient Temperature 406 Anti-Solvent (Ethanol) DMF: 30% H₂0 NoSolid Addition (4° C.) 407 Anti-Solvent (Ethanol) DMF: 30% H₂0 Form IIAddition (−21° C.) 408 Temp. Cycling 1,4-dioxane: 1% H₂0 Form II 409Controlled Cool (4° C.) 1,4-dioxane: 1% H₂0 No Solid 410 Controlled Cool(−21° C.) 1,4-dioxane: 1% H₂0 No Solid 411 Evaporation 1,4-dioxane: 1%H₂0 No Solid 412 Anti-Solvent (IPA) 1,4-dioxane: 1% H₂0 No SolidAddition Elevated Temperature 413 Anti-Solvent (IPA) 1,4-dioxane: 1% H₂0No Solid Addition Ambient Temperature 414 Anti-Solvent (IPA)1,4-dioxane: 1% H₂0 No Solid Addition (4° C.) 415 Anti-Solvent (IPA)1,4-dioxane: 1% H₂0 No Solid Addition (−21° C.) 416 Anti-Solvent(Ethanol) 1,4-dioxane: 1% H₂0 No Solid Addition Ambient Temperature 417Anti-Solvent (Ethanol) 1,4-dioxane: 1% H₂0 No Solid Addition (4° C.) 418Anti-Solvent (Ethanol) 1,4-dioxane: 1% H₂0 No Solid Addition (−21° C.)419 Temp. Cycling 1,4-dioxane: 3% H₂0 Form II 420 Controlled Cool (4°C.) 1,4-dioxane: 3% H₂0 No Solid 421 Controlled Cool (−21° C.)1,4-dioxane: 3% H₂0 No Solid 422 Evaporation 1,4-dioxane: 3% H₂0 Form II423 Anti-Solvent (IPA) 1,4-dioxane: 3% H₂0 No Solid Addition ElevatedTemperature 424 Anti-Solvent (IPA) 1,4-dioxane: 3% H₂0 No Solid AdditionAmbient Temperature 425 Anti-Solvent (IPA) 1,4-dioxane: 3% H₂0 No SolidAddition (4° C.) 426 Anti-Solvent (IPA) 1,4-dioxane: 3% H₂0 No SolidAddition (−21° C.) 427 Anti-Solvent (Ethanol) 1,4-dioxane: 3% H₂0 NoSolid Addition Ambient Temperature 428 Anti-Solvent (Ethanol)1,4-dioxane: 3% H₂0 No Solid Addition (4° C.) 429 Anti-Solvent (Ethanol)1,4-dioxane: 3% H₂0 No Solid Addition (−21° C.) 430 Temp. Cycling1,4-dioxane: 10% H₂0 Form II 431 Controlled Cool (4° C.) 1,4-dioxane:10% H₂0 No Solid 432 Controlled Cool (−21° C.) 1,4-dioxane: 10% H₂0 NoSolid 433 Evaporation 1,4-dioxane: 10% H₂0 Form II 434 Anti-Solvent(IPA) 1,4-dioxane: 10% H₂0 No Solid Addition Elevated Temperature 435Anti-Solvent (IPA) 1,4-dioxane: 10% H₂0 No Solid Addition AmbientTemperature 436 Anti-Solvent (IPA) 1,4-dioxane: 10% H₂0 No SolidAddition (4° C.) 437 Anti-Solvent (IPA) 1,4-dioxane: 10% H₂0 No SolidAddition (−21° C.) 438 Anti-Solvent (Ethanol) 1,4-dioxane: 10% H₂0 NoSolid Addition Ambient Temperature 439 Anti-Solvent (Ethanol)1,4-dioxane: 10% H₂0 No Solid Addition (4° C.) 440 Anti-Solvent(Ethanol) 1,4-dioxane: 10% H₂0 No Solid Addition (−21° C.) 441 Temp.Cycling MeOH: 5% H₂0 Form II 442 Controlled Cool (4° C.) MeOH: 5% H₂0 NoSolid 443 Controlled Cool (−21° C.) MeOH: 5% H₂0 No Solid 444Evaporation MeOH: 5% H₂0 Form II 445 Anti-Solvent (IPA) MeOH: 5% H₂0Form II Addition Elevated Temperature 446 Anti-Solvent (IPA) MeOH: 5%H₂0 Form II Addition Ambient Temperature 447 Anti-Solvent (IPA) MeOH: 5%H₂0 Form II Addition (4° C.) 448 Anti-Solvent (IPA) MeOH: 5% H₂0 Form IIAddition (−21° C.) 449 Anti-Solvent (Ethanol) MeOH: 5% H₂0 No SolidAddition Ambient Temperature 450 Anti-Solvent (Ethanol) MeOH: 5% H₂0Form II Addition (4° C.) 451 Anti-Solvent (Ethanol) MeOH: 5% H₂0 Form IIAddition (−21° C.) 452 Temp. Cycling MeOH: 20% H₂0 No Solid 453Controlled Cool (4° C.) MeOH: 20% H₂0 Form II 454 Controlled Cool (−21°C.) MeOH: 20% H₂0 Form II 455 Evaporation MeOH: 20% H₂0 Form II 456Anti-Solvent (IPA) MeOH: 20% H₂0 Form II Addition Elevated Temperature457 Anti-Solvent (IPA) MeOH: 20% H₂0 Form II Addition AmbientTemperature 458 Anti-Solvent (IPA) MeOH: 20% H₂0 Form II Addition (4°C.) 459 Anti-Solvent (IPA) MeOH: 20% H₂0 Form II Addition (−21° C.) 460Anti-Solvent (Ethanol) MeOH: 20% H₂0 Form II Addition AmbientTemperature 461 Anti-Solvent (Ethanol) MeOH: 20% H₂0 Form II Addition(4° C.) 462 Anti-Solvent (Ethanol) MeOH: 20% H₂0 Form I/II Addition(−21° C.) 463 Temp. Cycling MeOH: 50% H₂0 Form II 464 Controlled Cool(4° C.) MeOH: 50% H₂0 Form II 465 Controlled Cool (−21° C.) MeOH: 50%H₂0 Form II 466 Evaporation MeOH: 50% H₂0 No Solid 467 Anti-Solvent(IPA) MeOH: 50% H₂0 Form II Addition Elevated Temperature 468Anti-Solvent (IPA) MeOH: 50% H₂0 No Solid Addition Ambient Temperature469 Anti-Solvent (IPA) MeOH: 50% H₂0 Form II Addition (4° C.) 470Anti-Solvent (IPA) MeOH: 50% H₂0 No Solid Addition (−21° C.) 471Anti-Solvent (Ethanol) MeOH: 50% H₂0 Form II Addition AmbientTemperature 472 Anti-Solvent (Ethanol) MeOH: 50% H₂0 Form I Addition (4°C.) 473 Anti-Solvent (Ethanol) MeOH: 50% H₂0 Form I/II Addition (−21°C.) 474 Temp. Cycling THF: 1% H₂0 Form II 475 Controlled Cool (4° C.)THF: 1% H₂0 No Solid 476 Controlled Cool (−21° C.) THF: 1% H₂0 No Solid477 Evaporation THF: 1% H₂0 Form II 478 Anti-Solvent (IPA) THF: 1% H₂0No Solid Addition Elevated Temperature 479 Anti-Solvent (IPA) THF: 1%H₂0 No Solid Addition Ambient Temperature 480 Anti-Solvent (IPA) THF: 1%H₂0 No Solid Addition (4° C.) 481 Anti-Solvent (IPA) THF: 1% H₂0 NoSolid Addition (−21° C.) 482 Anti-Solvent (Ethanol) THF: 1% H₂0 No SolidAddition Ambient Temperature 483 Anti-Solvent (Ethanol) THF: 1% H₂0 NoSolid Addition (4° C.) 484 Anti-Solvent (Ethanol) THF: 1% H₂0 No SolidAddition (−21° C.) 485 Temp. Cycling THF: 3% H₂0 Form II 486 ControlledCool (4° C.) THF: 3% H₂0 No Solid 487 Controlled Cool (−21° C.) THF: 3%H₂0 No Solid 488 Evaporation THF: 3% H₂0 No Solid 489 Anti-Solvent (IPA)THF: 3% H₂0 No Solid Addition Elevated Temperature 490 Anti-Solvent(IPA) THF: 3% H₂0 Form II Addition Ambient Temperature 491 Anti-Solvent(IPA) THF: 3% H₂0 No Solid Addition (4° C.) 492 Anti-Solvent (IPA) THF:3% H₂0 No Solid Addition (−21° C.) 493 Anti-Solvent (Ethanol) THF: 3%H₂0 No Solid Addition Ambient Temperature 494 Anti-Solvent (Ethanol)THF: 3% H₂0 No Solid Addition (4° C.) 495 Anti-Solvent (Ethanol) THF: 3%H₂0 No Solid Addition (−21° C.) 496 Temp. Cycling THF: 5% H₂0 No Solid497 Controlled Cool (4° C.) THF: 5% H₂0 No Solid 498 Controlled Cool(−21° C.) THF: 5% H₂0 No Solid 499 Evaporation THF: 5% H₂0 Form II 500Anti-Solvent (IPA) THF: 5% H₂0 No Solid Addition Elevated Temperature501 Anti-Solvent (IPA) THF: 5% H₂0 No Solid Addition Ambient Temperature502 Anti-Solvent (IPA) THF: 5% H₂0 Form II Addition (4° C.) 503Anti-Solvent (IPA) THF: 5% H₂0 No Solid Addition (−21° C.) 504Anti-Solvent (Ethanol) THF: 5% H₂0 No Solid Addition Ambient Temperature505 Anti-Solvent (Ethanol) THF: 5% H₂0 No Solid Addition (4° C.) 506Anti-Solvent (Ethanol) THF: 5% H₂0 No Solid Addition (−21° C.) 507 Temp.Cycling butan-1-ol: 1% H₂0 Form II 508 Controlled Cool (4° C.)butan-1-ol: 1% H₂0 No Solid 509 Controlled Cool (−21° C.) butan-1-ol: 1%H₂0 No Solid 510 Evaporation butan-1-ol: 1% H₂0 Form II 511 Anti-Solvent(IPA) butan-1-ol: 1% H₂0 No Solid Addition Elevated Temperature 512Anti-Solvent (IPA) butan-1-ol: 1% H₂0 No Solid Addition AmbientTemperature 513 Anti-Solvent (IPA) butan-1-ol: 1% H₂0 No Solid Addition(4° C.) 514 Anti-Solvent (IPA) butan-1-ol: 1% H₂0 No Solid Addition(−21° C.) 515 Anti-Solvent (Ethanol) butan-1-ol: 1% H₂0 No SolidAddition Ambient Temperature 516 Anti-Solvent (Ethanol) butan-1-ol: 1%H₂0 No Solid Addition (4° C.) 517 Anti-Solvent (Ethanol) butan-1-ol: 1%H₂0 No Solid Addition (−21° C.) 518 Temp. Cycling butan-1-ol: 3% H₂0Form II 519 Controlled Cool (4° C.) butan-1-ol: 3% H₂0 No Solid 520Controlled Cool (−21° C.) butan-1-ol: 3% H₂0 No Solid 521 Evaporationbutan-1-ol: 3% H₂0 Form II 522 Anti-Solvent (IPA) butan-1-ol: 3% H₂0 NoSolid Addition Elevated Temperature 523 Anti-Solvent (IPA) butan-1-ol:3% H₂0 No Solid Addition Ambient Temperature 524 Anti-Solvent (IPA)butan-1-ol: 3% H₂0 No Solid Addition (4° C.) 525 Anti-Solvent (IPA)butan-1-ol: 3% H₂0 No Solid Addition (−21° C.) 526 Anti-Solvent(Ethanol) butan-1-ol: 3% H₂0 No Solid Addition Ambient Temperature 527Anti-Solvent (Ethanol) butan-1-ol: 3% H₂0 No Solid Addition (4° C.) 528Anti-Solvent (Ethanol) butan-1-ol: 3% H₂0 No Solid Addition (−21° C.)529 Temp. Cycling butan-1-ol: 5% H₂0 Form II 530 Controlled Cool (4° C.)butan-1-ol: 5% H₂0 No Solid 531 Controlled Cool (−21° C.) butan-1-ol: 5%H₂0 No Solid 532 Evaporation butan-1-ol: 5% H₂0 Form II 533 Anti-Solvent(IPA) butan-1-ol: 5% H₂0 No Solid Addition Elevated Temperature 534Anti-Solvent (IPA) butan-1-ol: 5% H₂0 Form II Addition AmbientTemperature 535 Anti-Solvent (IPA) butan-1-ol: 5% H₂0 No Solid Addition(4° C.) 536 Anti-Solvent (IPA) butan-1-ol: 5% H₂0 No Solid Addition(−21° C.) 537 Anti-Solvent (Ethanol) butan-1-ol: 5% H₂0 No SolidAddition Ambient Temperature 538 Anti-Solvent (Ethanol) butan-1-ol: 5%H₂0 No Solid Addition (4° C.) 539 Anti-Solvent (Ethanol) butan-1-ol: 5%H₂0 No Solid Addition (−21° C.) 540 Temp. Cycling 1,4-dioxane Form I 541Evaporation 1,4-dioxane Form I/II 542 Anti-Solvent Addition 1,4-dioxaneNo Solid 543 Temp. Cycling 1-butanol Form I 544 Evaporation 1-butanolForm I/II 545 Anti-Solvent (Hexane) 1-butanol Form III Addition (4° C.)546 Temp. Cycling ethanol Form I 547 Evaporation ethanol Form II 548Anti-Solvent (Hexane) ethanol Form I Addition (4° C.) 549 Temp. Cyclingacetone Form I 550 Evaporation acetone Form II 551 Anti-Solvent (Hexane)acetone Form III Addition (4° C.) 552 Temp. Cycling benzonitrile Form I553 Evaporation benzonitrile Form II 554 Anti-Solvent (Hexane)benzonitrile Form II Addition (4° C.) 555 Temp. Cycling cyclohexane FormI 556 Evaporation cyclohexane Form II 557 Anti-Solvent (Hexane)cyclohexane No Solid Addition (4° C.) 558 Temp. Cycling DCM Form I 559Evaporation DCM Form II 560 Anti-Solvent (Hexane) DCM Form III Addition(4° C.) 561 Temp. Cycling DMSO Form I 562 Evaporation DMSO Form II/II563 Anti-Solvent (Hexane) DMSO No Solid/No Addition (4° C.) Solid 564Temp. Cycling EtOAc Form I 565 Evaporation EtOAc Form II 566Anti-Solvent (Hexane) EtOAc Form III Addition (4° C.) 567 Temp. CyclingHeptane Form I 568 Evaporation Heptane Form I/II 569 Anti-Solvent(Hexane) Heptane No Solid/No Addition (4° C.) Solid 570 Temp. CyclingIPA Form I 571 Evaporation IPA Form I/II 572 Anti-Solvent (Hexane) IPANo Solid Addition (4° C.) 573 Temp. Cycling IPA: Water (1%) Form I 574Evaporation IPA: Water (1%) Form II 575 Anti-Solvent (Hexane) IPA: Water(1%) No Solid/No Addition (4° C.) Solid 576 Temp. Cycling MeCN Form I577 Evaporation MeCN Form II 578 Anti-Solvent (Hexane) MeCN Form I/IIIAddition (4° C.) 579 Temp. Cycling MeCN: Water (1%) Form I 580Evaporation MeCN: Water (1%) Form I/II 581 Anti-Solvent (Hexane) MeCN:Water (1%) No Solid Addition (4° C.) 582 Temp. Cycling MEK Form I 583Evaporation MEK Form I/II 584 Anti-Solvent (Hexane) MEK Form IIIAddition (4° C.) 585 Temp. Cycling MeOAc Form I 586 Evaporation MeOAcForm II 587 Anti-Solvent (Hexane) MeOAc Form III Addition (4° C.) 588Temp. Cycling MeOH Form I 589 Evaporation MeOH Form I/II 590Anti-Solvent (Hexane) MeOH Form III Addition (4° C.) 591 Temp. CyclingMIBK Form I 592 Evaporation MIBK Form II 593 Anti-Solvent (Hexane) MIBKNo Solid Addition (4° C.) 594 Temp. Cycling Nitromethane Form I 595Evaporation Nitromethane Form II 596 Anti-Solvent (Hexane) NitromethaneForm I Addition (4° C.) 597 Temp. Cycling TBME Form I 598 EvaporationTBME Form II 599 Anti-Solvent (Hexane) TBME Form I Addition (4° C.) 600Temp. Cycling THF Form I 601 Evaporation THF Form II 602 Anti-Solvent(Hexane) THF Form I/III Addition (4° C.) 603 Temp. Cycling THF: water(1%) Form I 604 Evaporation THF: water (1%) Form I/II 605 Anti-Solvent(Hexane) THF: water (1%) No Solid Addition (4° C.) 606 Temp. Cyclingtoluene Form I 607 Evaporation toluene Form II 608 Anti-Solvent (Hexane)toluene Form III Addition (4° C.) 609 Temp. Cycling water No Solid 610Evaporation water Form I/II 611 Anti-Solvent (Hexane) water Form IIIAddition (4° C.)

Example 2: Intrinsic Dissolution Studies

The intrinsic dissolution rates for Forms I, II, and III were measuredat pH conditions of 1.0, 4.5 and 6.7. The results are reproduced belowin TABLE 6. In each case, complete dissolution was achieved in less than3 minutes. Surprisingly, a pH dependence was observed for Form II; withthe intrinsic dissolution rate increasing with the pH. In contrast,Forms I and III appear to dissolve at rates independent of pH.

TABLE 6 Calculated Intrinsic Dissolution Rates (mg/cm²/s) 1.0 4.5 6.7Form I 0.41 0.44 0.37 Form II 0.26 0.34 0.62 Form III 0.49 0.44 0.45

Example 3: Solubility Studies

The solubility of L-ornithine phenyl acetate was approximated accordingto methods disclosed above. 24 solvents systems were tested: 1,4dioxane, 1-butanol, ethanol, acetone, benzonitrile, cyclohexane, DCM,DMSO, EtOAc, Heptane, IPA, IPA (1% H₂O), MeCN, MeCn (1% H₂O), MEK,MeOAc, methanol, MIBK, Nitromethane, THF, THF (1% H₂O), Toluene andwater. L-ornithine phenyl acetate exhibited a solubility in water,whereas L-ornithine phenyl acetate was substantially insoluble in theremaining solvent systems.

Slurries of L-ornithine phenyl acetate in water were also prepared andthe slurry was filtered. The filtrate concentration was analyzed byHPLC, and the results show the solubility of L-ornithine phenyl acetateto be about 1.072 mg/mL.

HPLC determinations of solubility were also completed for five solvents:ethanol, acetone, methanol, DMSO and IPA. These results are summarizedin TABLE 7.

TABLE 7 HPLC Solubility Determinations Solubility Solvent (mg/mL) PeakArea Comments EtOH <0.0033 N/A Small peak Acetone 0 0 API content beyondthe lower limit of quantification (LLOQ) MeOH 0.0033 1906.75  Resolvedpeak DMSO >0.0033 N/A Shoulder on DMSO peak IPA 0 0 API content beyondthe LLOQ

These results indicate that both acetone and IPA are suitable asanti-solvents for precipitating L-ornithine phenyl acetate. In contrast,solvents with measurable solubility are less favorable for precipitatingcrystalline forms of L-ornithine phenyl acetate.

Finally, the solubility of L-ornithine phenyl acetate was determined invarious mixtures of IPA and water using HPLC. The results are shown inTABLE 8.

TABLE 8 HPLC Solubility Determinations (IPA/Water) % IPA Peak AreaSolubility (mg/mL) 100 0 0 90 295 0.0054 80 2634 0.0455 70 8340 0.1433

Example 4: Small-Scale Batch Process to Produce L-Ornithine PhenylAcetate

About 8.4 g (0.049 moles) of L-ornithine HCl was dissolved in 42 mL H₂Oand, separately, about 11.4 g of silver benzoate was dissolved in 57 mLDMSO. Subsequently, the silver benzoate solution was added to theL-ornithine HCl solution. Combining the two mixtures resulted in animmediate, exothermic precipitation of a creamy white solid (AgCl). Thesolid was removed by vacuum filtration and retaining the filtrate(L-ornithine benzoate in solution). 200 mL of IPA was added to thefiltrate and the mixture was cooled to 4° C. A crystalline solidprecipitated after about 3 hours (L-ornithine benzoate) which wasisolated by vacuum filtration. Yield: 60%

7.6 g (0.03 moles) of the L-ornithine benzoate was dissolved in 38 mLH₂O and about 4.4 g of sodium phenyl acetate was dissolved 22 mL H₂O.Subsequently, the sodium phenyl acetate solution was added to theL-ornithine benzoate solution and left to stir for about 10 minutesAbout 240 mL of IPA (8:2 IPA:H₂O) was added and the solution stirred for30 minutes before cooling to 4° C. A crystalline solid precipitatedafter about 3 hrs at 4° C. (L-ornithine phenyl acetate). The precipitatewas isolated by vacuum filtration and washed with 48-144 mL of IPA.Yield: 57%

Example 5: Large-Scale Batch Process to Produce L-Ornithine PhenylAcetate

Two separate batch of L-ornithine phenyl acetate were prepared asfollows:

About 75 Kg of L-Ornithine monohydrochloride was dissolved in 227 kg ofwater. To the resulting solution was added 102 Kg of silver benzoatedissolved in 266 kg of DMSO at room temperature within 2 hours.Initially, a strong exothermy was observed and the silver chlorideprecipitated. The receiver containing the solution was then washed with14 Kg of DMSO that was added to the reaction mass. In order to removethe silver chloride formed, the reaction mass was filtered over a lensfilter prepared with 10 kg of Celite and a GAF filter of 1 mm. Afterfiltration, the filter was washed with an additional 75 kg of water. Thereaction mass was then heated at 35±2° C. and 80 kg of sodium phenylacetate was added. At this point the reaction mass was stirred at 35±2°C. for at least 30 minutes.

In order to precipitate the final API, 353 kg of isopropyl alcohol wasadded to the reaction mass. The reaction mass was then cooled to 0±3° C.within 6 hours, stirred for 1 hour and then the product isolated in acentrifuge.

About 86 kg of finished wet produce was obtained. The product was thendried at 40±5° C. for about 6.5 to 8 hours to provide about 75 kg ofL-ornithine phenyl acetate. Yield: 63.25. TABLE 9 summarizesmeasurements relating to the final product.

TABLE 9 Analytical Results for Large-scale Batch Process Test Batch 1Batch 2 Purity 98.80%  98.74%  Benzoate 0.17% 0.14% Silver 28 ppm 157ppm Chloride  0.006%  0.005% Sodium  7 ppm  26 ppm Total Impurities0.17% 0.14% Physical Form Form II Form II

Example 6: Reducing Silver Content in L-Ornithine Phenyl Acetate

Batch 2 from Example 5 exhibited high amounts of silver (157 ppm), andtherefore procedures were tested for reducing the silver content. Ninetrials were completed; each generally including dissolving about 20 g ofL-ornithine phenyl acetate from Batch 2 into 1.9 parts water, and thensubsequently adding 10.8 parts IPA. A crystalline form was isolated at0° C. by filtration.

For four trials, 8.0 mg or 80 mg of heavy metal scavengers SMOPEX 102 orSMOPEX 112 were added to the aqueous solution and stirred for 2 hours.The scavengers failed to reduce the silver content below 126 ppm.Meanwhile, another trial applied the general conditions disclosed aboveand reduced the silver content to 179 ppm. In still another trial, theL-ornithine phenyl acetate was slurried in a solution of IPA, ratherthan crystallized; however this trial also failed to reduce the silvercontent below 144 ppm.

The last three trials included adding diluted HCl to the solution toprecipitate remaining amount of silver as AgCl. The precipitate was thenremoved by filtration before The three trials included adding: (1) 1.0 gof 0.33% HCl at 20° C.; (2) 1.0 g of 0.33% HCl at 30° C.; and (3) 0.1 gof 3.3% HCl at 20° C. The three trials reduced the silver content to 30ppm, 42 ppm, and 33 ppm, respectively, and each trial yielding greaterthan 90% L-ornithine phenyl acetate. Accordingly, the addition of HClwas effective in reducing the amount of residual silver.

Example 6: Process for Preparing L-Ornithine Phenyl Acetate without anIntermediate Salt

As a general procedure, L-ornithine hydrochloride was suspended in asolvent. After that the reaction mass was heated and a base, sodiummethoxide, was added. NaCl formed and was removed from the system byfiltration. The reaction mass was cooled and a molar equivalent ofphenyl acetic acid was added to the reaction mass in order to formL-ornithine phenyl acetate. The final product was isolated, washed anddried. A summary of the trial for this process is provided in TABLE 10.

TABLE 10 Process Trials Trial Base Eq. of Base Solvent 1 NaOMe 21% inMeOH 1.0 eq. MeOH 2 NaOMe 21% in MeOH 0.95 eq.  IPA 3 NaOMe 21% in EtOH1.0 eq. EtOH 4 NaOMe 21% in MeOH 1.0 eq. MeOH 5 NaOMe 21% in MeOH 1.0eq. MeOH w/IPA for precipitation 6 NaOMe 21% in MeOH 1.0 eq.Acetonitrile 7 NaOMe 21% in MeOH 1.0 eq. Water/IPA 8 NaOMe 21% in MeOH1.0 eq. Water/IPA 9 NaOMe 21% in MeOH 1.0 eq. n-butanol

The resulting L-ornithine phenyl acetate was found to exhibit highamounts of chloride (at least about 1% by weight), and presumablyinclude similar amounts of sodium. The yields were about 50% for Trials2, 4, and 5.

Example 7: Thermal Stability Studies of Forms I, II, and III

Samples of Forms I, II and III were stored at increased temperatures anddesignated conditions as outlined in TABLE 11. The vacuum applied 600psi to achieve the reduced pressure. The final compositions were testedby XRPD, NMR, IR and HPLC to determine any changes to the material.

Most notably, Form III does not transition to Form II under vacuum at120° C., but rather exhibits greater chemical degradation compared toForms I and II under these conditions. Meanwhile, Form III converts toForm II and exhibits substantial chemical degradation at 120° C. withouta vacuum.

Form I converted to Form II in all the trials, but most interestingly,Form I exhibits substantial chemical degradation at 120° C. without avacuum. Thus, the conversion from Form I does not exhibit the samechemical stability as Form II, which is surprising considering thematerial readily converts to Form II.

Form II was stable and did not chemically degrade in all of the trials.Thus, Form II is the most stable. Meanwhile, Form III is more stablethan Form I, but both forms exhibit substantial chemical degradation at120° C. without a vacuum.

TABLE 11 Thermal Stability Trials Initial Trial Form TemperatureCondition Period Results 1 Form I 80° C. no 7 days Form II, no vacuumdegradation 2 Form I 80° C. vacuum 7 days Form II, no degradation 3 FormI 80° C. no 14 days Form II, no vacuum degradation 4 Form I 80° C.vacuum 14 days Form II, no degradation 5 Form II 80° C. no 7 days FormII, no vacuum degradation 6 Form II 80° C. vacuum 7 days Form II, nodegradation 7 Form II 80° C. no 14 days Form II, no vacuum degradation 8Form II 80° C. vacuum 14 days Form II, no degradation 5 Form III 80° C.no 7 days Form III, no vacuum degradation 5 Form III 80° C. no 14 daysForm III, no vacuum degradation 6 Form I 120° C. no 7 days Form IIvacuum (>96% API) 7 Form I 120° C. vacuum 7 days Form II (>99.9% API) 8Form I 120° C. no 14 days Form II vacuum (37% API) 9 Form I 120° C.vacuum 14 days Form II (>96% API) 8 Form II 120° C. no 7 days Form IIvacuum (98.6% API) 9 Form II 120° C. vacuum 7 days Form II (98.7% API)10 Form II 120° C. no 14 days Form II vacuum (>95% API) 11 Form II 120°C. vacuum 14 days Form II (>95% API) 10 Form III 120° C. no 7 days FormII vacuum (<30% API) 11 Form III 120° C. vacuum 7 days Form III (>95%API) 12 Form III 120° C. no 14 days Form II vacuum (<30% API) 14 FormIII 120° C. vacuum 14 days Form III (88.8% API)

HPLC results for the trials exhibiting chemical degradation (e.g., Trial10 from TABLE 11) are summarized in TABLE 12. Each degraded materialexhibits common peaks at relative retention times (RRT) of 1.9, 2.2,2.4, and 2.7, which suggests a common degradation pathway for differentforms.

TABLE 12 HPLC Results for Degraded Samples Main Peak Retention Time(min) Degradation/Impuirty Peak(s) HPLC Form Timepoint RetentionRetention ID Sample ID Tested Stability Test (day) Time (min) % PeakArea Time (min) % Peak Area 39 W00045/45/3 III 120° C. ambient pressure7 2.857 35.786 6.763 6.103 7.582 45.161 42 W00045/45/6 III 120° C. undervacuum (ca. 600 psi) 7 2.787 88.885 7.598 9.389 51 W00045/45/1 I 120° C.ambient pressure 14 3.499 37.826 6.766 3.948 7.569 42.525 9.707 3.628 53W00045/45/3 III 120° C. ambient pressure 14 3.476 30.394 6.763 5.9757.583 56.459 56 W00045/45/6 III 120° C. under vacuum (ca. 600 psi) 143.400 87.389 7.555 11.500

Example 8: Oxygen Stability Studies of Forms I, II, and III

Samples of Forms I, II and III were stored in 100% oxygen environmentsfor 7 or 14 days and analyzed by NMR and IR. The results establish thatForms I and II show no signs of degradation after 14 days. Only IRresults were completed for Form III at 7 days, and these results confirmthere was no significant degradation. TLC results for all samplesindicated a single spot with similar Rf values.

Example 9: UV Stability Studies of Forms I, II, and III

Samples of Forms I, II and III were exposed to ultraviolet (UV)radiation for 7 or 14 days. A CAMAG universal UV Lampe applied radiationto the samples with setting of 254 mμ. NMR and IR results show nodegradation of Forms I and II after 14 days. Similarly, Form IIIexhibits no degradation after 7 days as determined by NMR and IR. TLCresults for all samples indicated a single spot with similar Rf values.

Example 10: pH Stability Studies of Forms I, II, and III

A slurry of Forms I, II and III were formed with water and the pH valueadjusted to either 1.0, 4.0, 7.0, 10.0, and 13.2. The slurries werestored for 7 or 14 days, and subsequently the solids were removed byfiltration. Form I converted to Form II in all of the samples. NMR andIR results show Forms I and II did not degrade after 14 days in thevaried pHs, and similarly HPLC results show about 98% purity or more forthese samples. Form III also exhibited no degradation after 7 daysaccording to NMR and IR results. HPLC tests show about 95% purity ormore; however IR results show Form III converted to Form II over the7-day test. TLC results for all samples indicated a single spot withsimilar Rf values.

Example 11: Compression Studies of Forms I, II, and III

Samples of Forms I, II and III were subjected to 3 tons of force using aMoore Hydraulic Press for about 90 minutes. The resultant tablet's mass,diameter and thickness were measured to determine the density. Thetablets were also analyzed by NMR and IR. Form I transitioned to acomposition of Form II with a density of 1.197 kg/m³. Form II did notexhibit a transition and had a final density of 1.001 kg/m³. Finally,Form III did not exhibit a transition and had a final density of 1.078kg/m³.

Example 12: Process for Producing L-Ornithine Phenyl Acetate Via anAcetate Intermediate

Dissolve 25 mg of L-ornithine HCl 5 vols of H₂O, and then add excessacetic acid (about 5 vols) to form a slurry. Subject the slurry totemperature cycling between 25° and 40° C. every 4 hours for about 3days. Add 1 equivalent of phenylacetic acid (with respect toL-ornithine) and stir for about 4-6 hrs (possibly heat). Use IPA as ananti-solvent, add enough to obtain a ratio of 70:30 (IPA:H₂O). Isolateby vacuum filtration and dry for about 4-8 hrs at 80° C. to remove anyresidual acetic acid.

What is claimed is:
 1. A process for preparing L-ornithinephenylacetate, comprising intermixing phenylacetic acid or a saltthereof with an L-ornithine acetate solution to form a reaction mixture;and isolating a composition comprising a crystalline form of L-ornithinephenylacetate from the reaction mixture.
 2. The process of claim 1,wherein the L-ornithine acetate solution is prepared by intermixing anL-ornithine salt with acetic acid in a first solvent.
 3. The process ofclaim 2, wherein the L-ornithine salt is L-ornithine halide salt.
 4. Theprocess of claim 3, wherein the L-ornithine salt is L-ornithinehydrochloride.
 5. The process of claim 2, wherein the molar ratio of theL-ornithine salt to acetic acid is at least 1:2.
 6. The process of claim2, wherein the first solvent comprises water.
 7. The process of claim 1,further comprising adding a second solvent to the reaction mixturebefore isolating the crystalline form of L-ornithine phenylacetate. 8.The process of claim 7, wherein the second solvent comprises isopropylalcohol (IPA).
 9. The process of claim 1, further comprisingrecrystallizing the isolated crystalline form of L-ornithinephenylacetate.
 10. The process of claim 1, wherein the crystalline formof L-ornithine phenylacetate exhibits an X-ray powder diffractionpattern comprising at least three characteristic peaks, wherein saidcharacteristic peaks are selected from the group consisting of peaks atapproximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8° and 24.1° 2θ.
 11. Theprocess of claim 10, wherein the crystalline form of L-ornithinephenylacetate exhibits an X-ray powder diffraction pattern comprisingcharacteristic peaks selected from the group consisting of peaks atapproximately 6.0°, 13.9°, 14.8°, 17.1°, 17.8° and 24.1° 2θ.
 12. Theprocess of claim 1, wherein the crystalline form of L-ornithinephenylacetate exhibits an X-ray powder diffraction pattern comprising atleast three characteristic peaks, wherein said characteristic peaks areselected from the group consisting of peaks at approximately 4.9°,13.2°, 17.4°, 20.8° and 24.4° 2θ.
 13. The process of claim 12, whereinthe crystalline form of L-ornithine phenylacetate exhibits an X-raypowder diffraction pattern comprising characteristic peaks selected fromthe group consisting of peaks at approximately 4.9°, 13.2°, 17.4°, 20.8°and 24.4° 2θ.
 14. The process of claim 1, wherein the crystalline formof L-ornithine phenylacetate exhibits an X-ray powder diffractionpattern comprising at least three characteristic peaks, wherein saidcharacteristic peaks are selected from the group consisting of peaks atapproximately 5.8°, 14.1°, 18.6°, 19.4°, 22.3° and 24.8° 2θ.
 15. Theprocess of claim 14, wherein the crystalline form of L-ornithinephenylacetate exhibits an X-ray powder diffraction pattern comprisingcharacteristic peaks selected from the group consisting of peaks atapproximately 5.8°, 14.1°, 18.6°, 19.4°, 22.3° and 24.8° 2θ.